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The Methods oj 
Microscopical Research 



DR. GRA 




THE LIBRARY 

OF 

THE UNIVERSITY 
OF CALIFORNIA 

PRESENTED BY 

PROF. CHARLES A. KOFOID AND 
MRS. PRUDENCE W. KOFOID 



THE METHODS OF MICROSCOPICAL 
RESEARCH. 



THE METHODS 

OF 

MICROSCOPICAL RESEARCH 



A PEACTICAL GUIDE TO MICEOSCOPICAL 
MANIPULATION 



SECOND EDITION 

MUCH ENLARGED AND IN GREAT PART RE-WRITTEN 



BY 

ARTHUR C. COLE, F.B.M.S, 



ALL BIGHTS RESERVED 



Xonfcon 

BAILLIEEE, TINDALL & COX 
20 & 21, KING WILLIAM STREET, STKAND, W.C. 

PARIS. MADRID. 

1895 



PEEFACE TO THE SECOND EDITION. 



" The Methods of Microscopical Research " 
formed a portion of the " Studies in Microscopical 
Science," a serial publication in four volumes, issued 
and edited by the author, and was embodied (as a 
treatise) with Yolume II. of that work. The recep- 
tion accorded to it induced the author to publish it 
as a separate work. The first edition was rapidly 
taken up, and the book soon became out of print 
and unobtainable. In response to a very general 
demand it is now re-issued, this (the second) edition 
being greatly enlarged and the work, indeed, almost 
entirely re-written. It embodies the full results of 
the author's thirty years' experience in microscopical 
work, all the formulas from his private note book, all 
his own special processes and methods of working, 
and all his simple inventions for perfecting the work 
and ensuring absolute cleanliness and permanence 
in preparations. Nothing has been concealed, every- 
thing has been fully described and explained, and 
so plainly that the author entertains the hope that 
the treatise will be found really practical, and so will 
enable the student and the amateur microscopist 
to make preparations worth studying and retaining 
as cabinet specimens. 



PEEFACE TO THE SECOND EDITION. 



" The Methods of Microscopical Research " 
formed a portion of the " Studies in Microscopical 
Science," a serial publication in four volumes, issued 
and edited by the author, and was embodied (as a 
treatise) with Volume II. of that work. The recep- 
tion accorded to it induced the author to publish it 
as a separate work. The first edition was rapidly 
taken up, and the book soon became out of print 
and unobtainable. In response to a very general 
demand it is now re-issued, this (the second) edition 
being greatly enlarged and the work, indeed, almost 
entirely re-written. It embodies the full results of 
the author's thirty years' experience in microscopical 
work, all the formulae from his private note book, all 
his own special processes and methods of working, 
and all his simple inventions for perfecting the work 
and ensuring absolute cleanliness and permanence 
in preparations. Nothing has been concealed, every- 
thing has been fully described and explained, and 
so plainly that the author entertains the hope that 
the treatise will be found really practical, and so will 
enable the student and the amateur microscopist 
to make preparations worth studying and retaining 
as cabinet specimens. 



CONTENTS. 



INTRODUCTION. 

PAGE 

1. On Instruments and their Use 1 

2. On Reagents, their Constitution and Action 4 

3. On the Methods of Preparation 5 

4. On Microscopical Art ... ... ... ... ... 7 

CHAPTER I. 

The Microscope ...^ 10 

The Human Eye 13 

CHAPTER II. 

Instruments and their Use 27 

CHAPTER III. 

The Preparation of Animal Tissues 38 

CHAPTER IV. 

On Injecting Blood Vessels, &c 69 

CHAPTER V, 

On Staining Fluids and Staining 77 

On Staining Bacilli and Bacteria ... 91 



viii. Contents. 



PAGE 



Clearing Media ... .., ... ... ... ... 95 

Mounting Media and Cements 98 

Embedding and Freezing ... ... ... ... ... 109 

On Section Cutting and Microtomes ... ... ... 119 

CHAPTER VI. 

On Mounting 132 

On the Preparation and Mounting of Insects ... ... 138 

On the Preparation and Mounting of Vegetable Sections 141 

On the Preparation and Mounting of the Diatomacese ... 144 

t 

CHAPTER VII. 

On Microscopical Drawing and Painting 161 

CHAPTER VIII. 

On Photo-Micrography 180 



THE METHODS 



OF 



MICROSCOPICAL EESEABCH, 



INTRODUCTION. 



I. On Instruments and their Use. 

THE investigation of minute structure, whether 
organic or inorganic, can be exhaustively and satis- 
factorily accomplished only by the assistance of 
adequate optical instruments. The organ of vision 
in man is so constructed that the distance of most 
distinct vision alternates between eight to ten 
inches, and the intermediates between these dis- 
tances, from the normal eye of an adult. This is 
due to the alterable curvature of the crystalline lens 
which allows the practically parallel pencil of rays 
from a distant object, or the divergent rays from an 
object close to the eye, to be accurately focussed on 
the retina, or sensitive portion of that organ ; but if 
the object is brought still closer to the eye than the 
normal distance of most distinct vision, it gradually 
1 



2 METHODS OF MICROSCOPICAL EESEAECH. 

loses its power of accommodation in exerting the 
strain required to render the crystalline lens suffi- 
ciently convex to focus the image of the object 
upon the retina; about two inches from the eye 
this action becomes impossible. Within the limits 
of accommodation megascopical characters only can 
be appreciated. If the eye were capable of altering 
the curvature of its crystalline lens indefinitely, its 
power of vision would become illimitable, and tele- 
scopes and microscopes and other such instruments 
would be found unnecessary. But the bounds of 
natural limitation can be conquered by artificial 
means, and the interposition of a sufficiently convex 
lens between the near object and the eye alters the 
direction of the rays of light which proceed from 
the object so as to bring them within the scope of 
natural vision, and herein lies the theory on which 
the microscope has been constructed. The nearer 
an object approaches the eye, the greater does its 
visual angle (or angle produced by the intersection 
of rays, or straight lines, from the extreme points 
of the object) become, and, consequently, a larger 
image is focussed upon the retina. 

Optical instruments, therefore, are required on 
the very threshold of our " Studies " to enable us 
to enter the domains of histology and microscopical 
investigation, and they may be employed directly in 
a multitude of instances when the objects are of 
minute size. Equally numerous, however, are the 
substances and organisms which cannot be thus 
directly examined, but which require to be sub- 
jected to special processes and manipulation in 
order to render them suitable for microscopical 
examination. In the inorganic kingdom some 



INTBODUCTION. 3 

minerals, rocks, and chemical substances, elemen- 
tary and otherwise, are found in Nature in such 
minute particles as to render them suitable for 
immediate examination and study under the 
microscope, but, in the majority of instances, it 
is necessary either to pulverise, grind them down 
into the thinnest possible sections or sheets, or 
to precipitate them by chemical means ere they can 
be brought within the range of observation, and 
thus the petrologist must be provided with chisels, 
hammers, files and stones for grinding and polish- 
ing, and a variety of instruments adapted to the 
collection and subsequent treatment of specimens, 
whilst the chemist will require blow-pipes, test 
tubes, re-agents and balances, &c., &c. For 
investigations in the organic world, the vegetable 
histologist must provide himself with a vasculum, 
a trowel, dipping bottles, and all necessary col- 
lecting apparatus of all kinds, and' with all the 
various instruments necessary to facilitate the 
examination of the unicellular and more delicate 
forms of plant life, whilst the more highly organised 
and complex examples will require dissection or 
sectionising and, in many cases, chemical treatment 
before they can be examined to any useful purpose. 
The animal histologist again will find his work 
inextricably connected with that of his brother 
botanist, and, to a large extent, will work hand in 
hand with him, but, as his observations and 
inquiries progress, there will arise necessities for 
a collection of tools including scalpels, scissors, bone 
forceps and saws, needles, forceps of different sizes 
and forms, section knives, as well as the all-impor- 
tant and indispensable microtome ; whilst both 



4 METHODS OF MICROSCOPICAL RESEARCH. 

students will require all the staining re- agents and 
mounting media necessary for the proper treatment 
and permanent preparation (as slides) of both 
vegetable and animal specimens, tissues and sec- 
tions. 

The study of both organic and inorganic histology 
is so complicated, that verbal descriptions, however 
exhaustive and perfect, are, without microscopical 
demonstration, in many cases, wholly inadequate; 
diagrams and drawings, therefore, are constantly 
employed to supplement the descriptions of the 
lecturer. In the preparation of such valuable aids 
to verbal description, the microscope is a most 
valuable, if not an indispensable adjunct, and it 
has become necessary, equally for the student, 
the investigator, the observer, the demonstrator 
and the lecturer, to thoroughly understand the 
construction and the use of the microscope itself, 
and of the various apparatus necessary for careful 
observations and investigations. 



II. On Re=agents, their Constitution 
and Action. 

This is one of the most important branches of 
Microscopical Technology, and as staining is an 
art, so a thorough knowledge of the behaviour of 
the various tissues under the action of chemical 
re-agents is an absolute necessity alike for the 
observer and the student. It will be continually 
found that the optical -means at the disposal of the 
histologist are insufficient or inadequate to the 
resolution of structure, and although much may be 



INTRODUCTION. 5 

done with carefully directed and modified light and 
an equally careful arrangement of magnifying 
power, yet the utility of the judicious application 
of re-agents requires only to be understood to be 
appreciated. The knowledge of the chemical con- 
stitution, physical properties and modes of manu- 
facture of re-agents, often leads to the discovery 
of their specific action on minute and ultimate 
structure, whether these may consist in a revelation 
of inherent qualities or in a modification of details. 
It thus becomes incumbent upon the histologist 
to most carefully scrutinise the action of every 
description of re-agent, and equally carefully and 
systematically to record the results of such ex- 
aminations. 

Re-agents may be used in a variety of ways for 
the elucidation of structure and the detection of 
the constitution of tissues and bodies. Some are 
employed as chemical tests, others as staining 
media to differentiate the tissues or to induce 
chaoges and appearances whereby certain proper- 
ties are revealed, whilst others, again, are used as 
preservative media. To know exactly what to 
apply and how to apply it for the revelation of 
specific phenomena, and to obtain the best results 
is of the very essence of microscopical technique 
and manipulation. 

III. On the Methods of Preparation. 

In order to arrive at a correct knowledge of, and 
as a means to their study, both organic and in- 
organic specimens and the great majority of or- 
ganisms require preparation by special methods. 



6 METHODS OP MICROSCOPICAL RESEARCH. 

Thus, the processes of pulverisation, levigation, 
sectionising, and grinding of bones, teeth, minerals, 
and rocks are beset by difficulties of detail which 
prove to be insurmountable obstacles to the tyro 
until he shall have acquired the necessary know- 
ledge and skill, under which they to a great extent 
disappear, or are more or less easily surmounted. 
So, also, want of success in section-cutting, stain- 
ing and mounting specimens is referable to the 
ignorance or neglect of minute precautions, to the 
want of absolute cleanliness in all processes, and 
the absence of the delicacy of manipulation without 
which success is impossible of attainment. In the 
opening pages of " How to Work with the Micro- 
scope," 1 Dr. Beale makes the following observa- 
tions : " Manual dexterity, although subordinate 
to many higher mental qualifications, is as essential 
for the successful prosecution of microscopic obser- 
vation as it is for that of every kind of experi- 
mental science. It assists us in the discovery of 
new means of inquiry and in devising methods by 
which difficulties may be surmounted. Without 
skilful manipulation we can neither teach by 
demonstration facts which have been already dis- 
covered, nor hope to extend the limits of observa- 
tion and experimental knowledge. It is not, 
therefore, surprising that many of the most impor- 
tant facts which have been recently added to 
microscopical science, have been discovered by men 
who had previously well-trained themselves in ex- 
periment particularly in practical chemistry and 
minute anatomical dissection. Improvements in 

1 Fifth edition, London, 1880, p. 1. 



INTRODUCTION. 7 

the practical details of manipulation almost neces- 
sarily precede an advance in natural knowledge, 
and invariably promote and expedite true scientific 
progress." 

But although manipulative skill is a very necessary 
adjunct to microscopical research, an attainment of 
the understanding of the general principles of action 
at the outset, sometimes proves to be the most 
arduous portion of the work, and very often is the 
only impediment to success. Practice and perse- 
verance, brought to bear upon previously gained 
knowledge, are the only royal roads to manual 
dexterity, and it thus becomes the duty of the 
instructor to point out, not merely what path 
ought to be taken, but the various pit-falls which 
everywhere surround the beaten track, and how 
best to avoid them. 

IV. On Microscopical Art. 

There are two ways in which microscopical 
objects can be drawn so as to become useful records 
of research. By the first of these, a rough dia- 
grammatic representation may be made, without 
reference to accuracy of form or size ; merely to 
display the observer's views concerning the struc- 
ture of the object. The second method is to make 
an accurate drawing with due regard to the size and 
form of the object under the microscope. Both of 
these methods are valuable in themselves, but their 
usefulness becomes immeasurably enhanced when 
they are combined so as to afford scope to the 
artistic skill and scientific knowledge of the 
draughtsman. 



O METHODS OF MICROSCOPICAL RESEARCH. 

There is a great deal of truth in the statement 
that true art is the outcome of genius, but that does 
not in any way affect microscopical drawing ; it 
will be found that patience and practice are suffi- 
cient to enable the student to overcome every 
obstacle, and to achieve the most satisfactory results 
in this department of art. 

But whilst admitting the perfection to which 
many great artists in microscopical drawing have 
attained, and the great assistance to microscopical 
research afforded by the works of those who, in 
addition to correctness of vision and observation, 
possess an educated eye and a hand capable of 
drawing exactly what they see, give us the benefit 
of exquisite colouring in addition to correctness of 
outline and detail, who can sufficiently estimate the 
incalculable advantages bestowed upon microscopical 
science by micro-photography, which producing 
pictures absolutely faultless and free from such 
slight defects as are inseparable from drawings 
which, being dependent for their production upon 
human eyes, brain and nerves, must inevitably be 
liable to human imperfections, whilst a micro-photo- 
graph from the hands of a skilled observer and 
photographer is as inevitably free from all errors 
and defects, and provides for us an actual and per- 
fect enlargement in all its features and details 
colour alone excepted of the image focussed upon 
the plate ? Micro-photography has indeed proved a 
boon to the microscopist, and the improvements 
which have been made and are daily being intro- 
duced in both apparatus and processes entitle us to 
believe that valuable and exquisite as are the 
pictures now produced by photography, the future 



INTRODUCTION. 9 

has greater and even more wonderful results in 
store for us. It is indeed impossible to overrate 
the benefits conferred alike upon science, the 
student, the teacher and the lecturer by this invalu- 
able art. 



10 



CHAPTER I. 



On Instruments and their Use: 
The Microscope. 

THE microscope, as an instrument of power in 
histological research, depends essentially in con- 
struction, upon its conformity with the laws of light 
and human vision. It has already been stated that 
an idea of the size of an object is arrived at through 
the size of the image focussed upon the retina, and 
that these dimensions vary in proportion as the 
object is brought near to, or removed farther from, 
the eye. When the object is brought close to the 
eye, its visual angle that is, the angle formed by 
the crossing of the rays from the extreme points of 
the object, is larger than when it is placed farther 
off, and, consequently, the image on the retina is 
larger also. If this principle were capable of un- 
limited extension, it would obviously follow, that to 
continue to increase the magnification of an object, 
all one would have to do would be to bring the 
object closer and closer to the eye. But there is 
a limit to this natural power of microscopical vision 
in the human subject, and the eye fails signally to 
accomplish its office when the object is brought 



ON INSTRUMENTS AND THEIR USE. 11 

within about two inches from its surface. The 
reason of this is, that the crystalline lens of the 
eye, in assuming a more convex shape through the 
relaxation of the ciliary muscle, becomes overtaxed 
at this distance. If, now, a sufficiently convex lens 
be placed between the object and the eye, so as to 
enable the divergent rays to be accurately focussed 
upon the retina, the difficulty will be overcome, 
and, theoretically, microscopical vision would be 
illimitable. But, is it so ? Most certainly not. 
The employment of artificial substances, such as 
crown and flint glass, diamonds, &c., although they 
considerably extend the power of sight, do not do 
so ad infinitum. Here the limitation is purely 
material, as distinguished from the former instance, 
the human eye, which is defective not only materi- 
ally, but physiologically. 

The worker in the field of microscopical research 
need not, however, be appalled by these statements, 
for it will be found that the human organ of vision, 
in conjunction with the excellent appliances of 
modern invention, will enable him to approach, 
and sometimes even to solve satisfactorily, many of 
those philosophical problems which underlie the 
evolution of things, both animate and inanimate. 
In exemplification of this, a few instances may here 
be recorded. 

The practical geologist who sallies forth into the 
field with lens in hand, may gather during his walk 
a variety of rocks, which, from their cosmological 
structure, point to an igneous origin ; some of the 
specimens are coarse grained, whilst others defy the 
utmost scrutiny of the eye. The microscope is 
brought to bear upon the question, and he finds 



12 METHODS OF MICROSCOPICAL RESEARCH. 

that a power of 500 diameters is generally the 
utmost degree of amplification he will require to 
employ ; but that for all practical purposes powers 
of from 20 to 100 diameters suffice. "With the 
assistance of the microscope he is enabled to pro- 
nounce with decision that the rocks are igneous ; 
and more, from analytical and synthetical experi- 
ments he can show that certam coarse varieties, 
which are thoroughly crystalline (the crystals being 
simply held together by adhesive and cohesive 
forces, without the necessity of an interstitial bind- 
ing substance), are of deep-seated origin, and con- 
solidated under conditions of enormous pressure 
and length of time. He is, in like manner, able to 
affirm of the other varieties, what their mineral 
constituents are, or have been, and how they came 
to assume their present states. Thus he builds a 
part of the fabric of geological philosophy, and with 
what ? with a comparatively low power of the 
microscope. 

To take an example from the organic world ; the 
questions of the function of various parts of the 
body, are very often arrived at through a minute 
study of its members. The form and general ap- 
pearance of the cells of glands such as the salivary 
glands, point to the functions they perform, whether 
they secrete or absorb, and how and when they 
perform their duties. The study of amoeboid, and 
even of ciliary motion, under the microscope, does 
not require a power of magnification much beyond 
700 diameters, whilst the life history of the minute 
forms of life known as germs (bacteria, fyc.), may 
be readily comprehended by the use of from 700- 
1,200 diameters. It is only when such things as the 




THE HUMAN EYE. 



ON INSTRUMENTS AND THEIR USE. 13 

delicate markings on the siliceous valves of Diatoms, 
or the artificial ruled lines on glass (Nobert's test) 
require to be made out, that powers beyond 1,200 
diameters become useful ; and it must be admitted 
that the scientific investigator does not lose any- 
thing, nor are any of his philosophical deductions 
vitiated by eschewing such powerful instruments, 
which, in the hands of the skilful, succeed in 
amplifying and resolving certain pretty structures ; 
but, to the ordinary worker are, in reality, impedi- 
ments to research. 

On the threshold of this inquiry, it thus becomes 
evident, that an acquaintance with the general 
structure of the human eye, coupled with the 
principles of luminous energy, are necessary adjuncts 
to an understanding of the microscope, its use and 
power. 

The Human Eye. 

DESCRIPTION OF PLATE. 

&, Anterior chamber of eye filled with the 
aqueous humour. 

c, Cornea, i, iris, p, pupil. 

cm, Ciliary muscle, cp, ciliary processes. Z, lens. 

s, Sclerotic coat, c, choroid. r, retina. 

/, Fovea. RR, superior and inferior recti muscles. 

0, Optic nerve entering the sclerotic and choroid 
coats. 

v, Vitreous humour. 



In all studies, whether of pure Microscopy as a 
Science, or whether of one of those departments of 
natural history in which the microscope is employed 



14 METHODS OF MICROSCOPICAL RESEARCH. 

as an aid to vision, we must, at the outset, recognise 
the importance of a study of the human eye. 

It may be the seat of many imperfections result- 
ing from misuse, old age, or disease, which are apt 
to modify the conclusions we may draw from our 
observations, unless we are careful to study well 
into what lines such imperfections may lead us. 

Nature has given us in this organ a means 
whereby all objects may be compared with each 
other, more especially as to size, colour, and general 
characters, and it must astonish the student, who 
thinks deeply, to find that so little is known definitely 
as to how we are able to appreciate magnitudes, 
colours, and forms. It is easy to say that the lenses 
of the eye focus a picture of the object upon the 
retina, and the irritations are carried by the optic 
nerve to the brain, but do we practically realise 
what this means ? 

Then, again, unless more of our senses than one 
are brought to bear upon a matter under considera- 
tion, we can scarcely form a true opinion upon our 
subject. 

Take something which greets our vision for the 
first time. We know not what it is ; we can see 
it, it is true, but we have to bring in the aid of 
other senses before we can arrive at a correct 
judgment; and even then, our judgment being the 
result of comparison, and also of experimental 
contact of substances with our senses so to speak 
opinions which are formed must, to a certain 
extent, be modified by the amount of other 
experience to which our nerve centres have been 
previously subjected. 

Take two experts; give to each one a sphere 



ON INSTRUMENTS AND THEIR USE. 15 

composed of lead and tin. Upon asking them what 
substance they were handling they might probably 
guess, perhaps not ; they would poise it in their 
hands, look at it, smell it, try to cut it, perhaps, 
examine its metallic lustre, and it would be very 
odd indeed if they could agree as to the composition 
of the alloy, unless settled by an assay upon the 
balance. 

Has it ever occurred to the reader that such pro- 
cesses as these go on in Microscopy, and that it is 
necessary to carefully study the organ of vision in 
order to gain a true insight into the object presented 
to us ? On reference to plate 1, it will be seen that 
the eye is a nearly spherical ball, capable of many 
movements in its socket. It possesses an outer 
translucent covering called the sclerotic coat (or 
simply sclerotica) which may be seen at S. This is 
thick, horny, and opaque, except in its anterior 
portion. 

This sclerotic coat envelopes about f of the eye- 
ball, and in common parlance is called the white of 
the eye. 

The anterior transparent portion is called the 
cornea, and has the shape of a very convex watch 
glass. It is through this membrane that the light 
passes to the interior of the eye. The cornea and 
the interior portion of the sclerotica are covered 
with a mucous membrane. 

Behind the cornea is a diaphragm of annular form 
called the iris ; it is coloured and opaque, the 
circular aperture in its centre, p, being called the 
pupil. 

The iris, i 9 serves the purpose of regulating the 
admission of light ; it varies in colour in different 



16 METHODS OF MICROSCOPICAL RESEARCH. 

individuals, and is the part referred to when we 
speak of the colour of a person's eye. 

Behind the pupil is the crystalline lens, I, having 
a much greater convexity at its posterior surface 
than at the anterior. 

The large posterior chamber is lined by the 
choroid coat, and this choroid has in front of it a 
delicate membrane called the retina. 

The choroid coat consists of a highly vascular 
membrane containing pigment cells, filled with an 
intense black mucus, called the pigmentum nigrum. 

The cavity behind the cornea is filled with a 
liquid called the aqueous humour, having a refractive 
index approaching that of 1*3366, while the larger 
cavity is filled with a transparent jelly, called the 
vitreous humour, possessing a refractive index of 
1*3379, enclosed in a very thin, transparent sac, 
called the hyaloid membrane. 

I have now described the principal apparatus of 
the eye, and may take some of the parts in detail. 

The crystalline lens is built up of layers, increas- 
ing in density inwards, the effect of which is to 
diminish spherical aberration. This lens is enclosed 
in a transparent capsule, held in position by an elastic 
membrane. It can be changed in shape by means 
of a delicate muscular arrangement to adapt its 
focus for near or distant objects. 

As glass lenses of varying curves have different 
focal lengths, so by altering the curves of the 
crystalline lens we are able to see objects distinctly 
which are situated in several focal planes. 

The reader may have noticed that there is a near 
point at which objects can be seen most distinctly ; 
this point varies in individuals, but averages from 



ON INSTRUMENTS AND THEIR USE. 17 

8 to 10 inches. As we move farther away from 
the object, although diminished in size, it may be 
seen more easily, and with less effort. 

It would appear, then, that all objects are ren- 
dered apparently larger, as they continue to 
approach the eye, but a limit is soon found to this, 
as at a distance of six inches distinct and easy 
vision is not possible (except in very abnormal 
cases). 

The reason of this is well-known the anterior 
focal point of a convex lens when shortened 
lengthens the posterior conjugate focus, so that 
when an object is brought too near the eye the 
image of it is projected behind the retina, and the 
crystalline lens cannot accommodate itself to such 
extremes. But we know that objects can be seen 
distinctly at great distances apart, and it may be 
useful to demonstrate how this is brought about. 

The real mechanism of accommodation has been 
much disputed, but the results, as observed, are, 
that the curvatures of the crystalline lens are 
altered as the observer adapts his eye to near or 
remote vision ; increase of curvature, of course, 
shortening the focal length of the crystalline lens, 
and being better adapted for near vision, while the 
shallower curve is necessary for the distant view of 
remote objects. Helmholtz has shown that the 
radius of curvature of the anterior surface of the 
crystalline lens may be varied by means of the 
muscular arrangement, from 6 to 10 millimetres. 

We may now cast another glance at the iris. 

This apparatus is really a continuation of the 

choroid tunic which lies between the sclerotica and 

the retina : it ends in front, in what are called 

2 



18 METHODS OF MICROSCOPICAL RESEARCH. 

ciliary processes. The small muscular ring sur- 
rounding the pupil is called the sphincter muscle. 

Now, the principal use of the choroid tunic, or 
rather the pigmentum nigrum which it contains, is 
to absorb those rays of light which have passed 
through the transparent retina, preventing their 
reflection, which would interfere with the distinct- 
ness of the image. 

By reference to the plate it will be seen that the 
choroid tunic, the retina, and sclerotica form the 
three outer rings, while the centre is ramified by 
nerve filaments and blood-vessels. 

These nerve filaments and blood-vessels lie in the 
retina, which really forms a continuation and exten- 
sion of the optic nerve ; it touches the outer cir- 
cumference of the iris at the front, and lies open as 
a cup-shaped disc in the interior of the eye ; it 
receives the rays of light which have passed in turn 
through the cornea, aqueous humour, crystalline 
lens, and vitreous humour, and forms a picture at 
the focus of these. 

The nerve fibres of the retina are excited 
probably by a product of the action of the light 
picture upon the visual purple, and the irritations 
are transmitted to the brain by the optic nerve, 
producing the sensation of vision. 

The picture produced upon the retina has been 
compared with that produced by a photographic 
lens upon a screen or ground glass ; but it will be 
seen that the instances are not strictly parallel. 

In the eye the rays falling upon the cornea do 
not again encounter air, the picture is formed in 
the highly refractive substance, while in the photo- 
graphic image air intervenes between the screen 
and the lens, and between the lenses themselves. 



ON INSTRUMENTS AND THEIE USE. 19 

Then, again, the adaptation of the eye to various 
distances is obtained by a process so dissimilar to 
that of the lens in the camera, that it is well no 
comparison should be instituted. 

The retina has been previously described as a 
delicate membrane lining the choroid tunic, inside 
the sclerotica. 

Now, if we make a section of the retina, and 
examine it under the microscope we shall find its 
structure to be as follows. 

Starting from the junction of the retina with the 
vitreous humour, we have : 

(1) The layer of nerve fibres. 

(2) The layer of nerve cells. 

(3) The granular layer. 

(4) The inner granular layer. 

(5) The intermediate layer. 

(6) The outer granular layer. 

(7) A second fine membrane. 

(8) The layer of rods and cones. 

(9) Pigmentum nigrum of the choroid. 

The retina is the terminal organ of vision, all the 
apparatus in front of it being merely for the 
purpose of securing that an accurate image shall 
be focussed upon it. As to how the luminous im- 
pressions yield to us such a definite idea of things 
is a question still under consideration ; many have 
tried to solve it, but it is open to doubt whether we 
are any nearer the mark than those philosophers 
who lived 2,000 years ago. 

There are several curious properties inherent in 
the retina. By means of the ophthalmoscope may 
be seen a point, a little out of the centre, where the 
optic nerve enters the eye. This spot is totally 



20 METHODS OF MICROSCOPICAL RESEARCH. 

blind, it cannot perceive a trace of light, and if 
the image of an object falls upon this blind spot, 
that object is totally invisible e It is at this spot 
also where the blood-vessels enter the eye, and 
ramify through nearly the whole of the surface 
layers of the retina. 

In the above description points only have been 
touched which directly bear on good or defective 
vision. On the other hand, enough has been 
advanced to show that this organ is liable to im- 
perfections which may be, and are, extremely liable 
to modify all our observations made over the tube 
of the microscope. 

Now, if we take an ordinary lens of glass and 
attempt to produce a picture with it, we find the 
centre alone is plainly visible the lens is afflicted 
with what is termed spherical aberration, that is, 
the rays from its periphery are brought to a focus 
in a different plane to those occupying a central 
position. 

But a small amount of spherical aberration is not 
readily detected by the student. It appears as a 
haze or fog of light over the object. 

In the human eye this defect is not observable to 
any great degree, as the peripheral or more strongly 
refracting rays are cut off by the iris. Then, again, 
the curvature of the cornea is ellipsoidal rather than 
circular, so that the rajs farthest from the axis are 
least deviated, while the two curves of the crystal- 
line lens correct, so to speak, the one the other ; 
and lastly, this lens is of such construction that its 
refractive power diminishes from the centre to the 
circumference. 

Another defect in the eye is due to the different 
meridians having dissimilar degrees of curvature. 



ON INSTRUMENTS AND THEIE USE. 21 

If a set of concentric circles be observed with one 
eye, they are seldom all distinct at the same time, 
and there is produced a kind of Maltese-cross effect, 
not perceivable, perhaps, in many instances with 
large circles, but noticeable when drawn to such 
a size that the outer one is about two inches in 
diameter. 

This defect is called astigmatism, and known to 
oculists as a common cause of headaches. Spasm 
of the focussing apparatus may derange the spheri- 
city of the eye, and so affect vision. Strained vision 
is very subject to this. On the other hand, the 
same apparatus may be paralysed, and ordinary 
vision deficient, whilst the focussing of the micro- 
scope might possibly correct it. 

Astigmatism has injuriously affected painters ; 
Turner, for instance, whose later pictures are dis- 
covered to be slightly distorted, in consequence of 
the power of accommodation or self -correction 
having been lost through age. 

In microscopic drawing, as with the camera 
lucida, the perspective may be misrepresented, in 
consequence of astigmatism, and thus endless dis- 
putes may arise even among the most careful 
observers. 

We have now to deal with errors of refrangibility, 
and it will probably have been assumed that the eye 
apparatus is entirely corrected for colour. This is 
not the case, however, except when an object is in 
exact focus, and the reason that the error due to 
refrangibility remains practically unnoticed is that 
the distance between the focal point of the red and 
violet rays is extremely small. The error due to 
refrangibility may be noticed by means of the con- 



22 METHODS OF MICROSCOPICAL RESEARCH. 

centric circles already referred to ; by bright day- 
light adjust the eyes to some object twelve inches 
away, and without moving the eye insert at a dis- 
tance of four inches a card inscribed with black 
circles, when a yellow and blue colouring will be 
plainly discerned. 

In order that the reader may thoroughly under- 
stand the error of refrangibility, the picture afforded 
by the passage of a solar ray through a prism of 
glass may be thrown upon a screen ; the rays are 
deflected unequally, the red least and the violet 
most. 

It may be advisable here to state that the degree 
of dispersion of the rays of white light depends 
upon the medium through which the ray passes, 
and this amount of dispersion is measured by the 
distance of the most prominent dark lines in the 
spectrum from each other. The diamond disperses 
much less than crown glass, while the deflection of 
the ray is greater ; but this is a subject beyond the 
scope of the present essay. 

Now, beside these errors, there are others to 
which the microscopist should devote special atten- 
tion ; they are caused by small opaque particles 
existing in the transparent media of the eye-ball. 
These cast their shadow on the retina, and produce 
images which appear to exist outside the eye. 
These extra-retinal images often appear as globules, 
bacteroid- shaped bodies, or strings of minute pearls, 
and may be studied by directing the eye to a sheet 
of strongly illuminated opal glass, through a small 
aperture made with a fine needle in a piece of thin 
blackened cardboard. 

When the microscope is used in a vertical position, 



ON INSTRUMENTS AND THEIR USE. 23 

these globules often gravitate to the centre of the 
cornea, and even after prolonged use of the inclined 
tube an observer may often be perplexed by the 
layer of mucus, or a lachrymal discharge covering 
the surface of the cornea. 

Colour is a special sensation excited in the retina 
by rays of a definite wave length, and the reason 
why certain objects are presented to our view with 
colour is that when white light falls upon a given 
surface, some is absorbed, the remainder being 
reflected. If the green rays are reflected, then the 
object appears green, and if the red rays are alone 
reflected, then the object will be red. 

The generally accepted theory of colour percep- 
tion is based on the assumption that three kinds of 
nerve fibres exist in the retina, the excitation of 
which produces sensations of red, green and violet, 
and that modifications of these three sensations 
yield all intermediate tints. 

This theory will explain some of the phenomena 
of colour blindness ; if the nerve fibres which give 
their special sensation are paralysed, or are absent, 
the sensation only of the complementary tint will 
be transmitted with all the defects of the eye. It 
must not be forgotten that many phenomena con- 
sist more in errors of judgment than in absolute 
error of form or sensation. 

Now in regard to errors of judgment, we must 
admit that all our estimations are made by compari- 
son. In magnitude we are guided by the size of 
the retinal image as determined by the visual angle 
for position we must have some starting point; 
and as for distance, every one knows how delusive 
an inexperienced estimate of this is. At sea, a 



24 METHODS OF MICROSCOPICAL RESEARCH. 

landsman could not judge of the distance of a pass- 
ing vessel to a few miles, nor could we form any 
accurate idea of the size of any object emitting 
practically parallel rays unless we had something 
to compare it with. 

We now come to a point which has been much 
disputed in the study of microscopy binocular 
vision. 

The two eyes move together as a system, so that 
we direct the two lines of regard to the same point 
in space and consequently see but a single image ; 
but it is possible to see two if one eye be displaced 
a little with the finger two images are seen, while 
if the other be displaced to a corresponding degree 
the one image is restored. 

The value of binocular vision may be easily 
ascertained by experiment. When a picture is 
presented to the retina of each eye, the compound 
picture is much brighter than when one retina only 
is employed. 

To each point of the retina of one eye there is a 
corresponding point in the retina of the other, and 
impressions produced on one of these points are in 
ordinary circumstances indistinguishable from similar 
impressions produced on the other. 

When both retinse are similarly impressed, the 
general effect is that the impressions are more 
intense than when one eye only is employed ; and 
we also get a perception of relief, that is of form in 
its three dimensions. 

Take two A eyepieces and look through them to 
the sky, so that two distinct circles are seen ; now 
bring them together so that one circle overlaps the 
other, when this overlapping bi-convex portion will 



ON INSTRUMENTS AND THEIR USE. 25 

be found double the brightness of the remaining 
portions of the circles. 

We are indebted to stereoscopic vision for the 
perception of relief or form in three dimensions, 
which occurs when the images falling upon the 
corresponding points of the two retinse are not 
exactly similar. In looking at an object with both 
eyes the rays do not run parallel from one side of 
the object to the eye on that side, but the right eye 
centres itself to the left side of the object and vice- 
versa. This may readily be seen by holding up a 
finger between our eyes and the wall, and looking at 
the latter. Two fingers may be seen projected on 
the wall, one of these is seen by the right eye and the 
other by the left ; but our visual impressions do not 
inform us which picture is formed by either eye in 
particular. Now, while steadfastly looking at the 
wall, close the right eye and the left finger will 
disappear, while on shutting the left eye, the right 
finger is rendered invisible. 

When two similar pictures are presented to the 
eyes, the impression is more vigorous and looked 
at with greater ease than when one eye only is 
employed; vision in this case is called pseudoscopic. 

Binocular vision should be employed wherever 
practicable ; it will be found much less trying to 
the eyes than monocular efforts. 

It has now been shown that the human eye is 
extremely liable to imperfections, and being so, strict 
attention to details is demanded from the micro- 
scopist. Now, although the human eye is such a 
wonderful instrument, there are many problems it is 
unable to solve without extraneous assistance. Take, 
for example, the bunt of wheat, Tilletia faries. With 



26 METHODS OF MICROSCOPICAL RESEARCH. 

the unaided eye, you will be able to discern nothing 
more than a black dust, the various details having to 
be made out by other means. Then again, with ob- 
jects so minute as the diatom, Amphipleura pellucida, 
the object itself is almost invisible to the unassisted 
eye, to say nothing of the beautiful carvings with 
which the valves are embellished, and which exact 
for their elucidation the most perfect lenses with 
which we are acquainted, and the most accurate 
manipulation of the illumination. You may, 
indeed, see the contour of many forms of diatoms 
without extra optical assistance than that afforded 
us by nature, but not much more than this, as if the 
eye is approached too closely the picture falls behind 
the retina and is lost. 

I have already mentioned the fact that starting 
with the distance of most distinct vision, continued 
approach to the eye finally renders the object in- 
visible, the rays being thrown behind the retina, the 
mechanism of accommodation being insufficient to 
produce a curve deep enough to bring the picture 
to a short conjugate focus. 

This can, however, be done by interposing a lens, 
or lenses, between the object and the cornea, so that 
a virtual image of the object is seen. These lenses 
form either a simple, or a compound microscope. 



27 



CHAPTER II. 

IN continuation of the subject of " Instruments 
and their Use," the author, holding in the highest 
estimation that invaluable treatise, Dr. Lionel S. 
Beale's " How to Work with the Microscope," as a 
thoroughly practical work, takes the liberty of 
quoting therefrom the following wise observations, 
which he considers cannot be too widely dis- 
seminated. " By describing the results of the in- 
vestigations of others, a teacher may spread 
knowledge. By prosecuting original enquiries 
himself, he may contribute his mite to the gradually 
increasing stock of information ; but by demon- 
strating to his pupils the successive steps by which 
conclusions in scientific enquiries have been at 
length arrived at, and by describing minutely the 
methods which have been actually employed in 
investigation, the teacher not only encourages his 
pupils to become original observers, and to investi- 
gate for themselves, but he may succeed in placing 
them in a position to commence their researches at 
the point where an enquiry has been abandoned by 
preceding observers. 

" The opinion that it is only necessary to place an 
object in the field of the microscope in order to 
make out its structure, seems far too prevalent. 



28 METHODS OF MICROSCOPICAL BESEARCH. 

Much of the disappointment suffered by many who 
are provided with microscopes may be traced to 
this erroneous idea. Too many look upon the 
microscope as a mere toy, and microscopical obser- 
vation as an amusement, by the help of which time 
may be made to pass away pleasantly. Few are 
aware of the real interest derived from intelligent 
investigation, and the instruction afforded, and the 
facts for contemplation and thought easily to be 
obtained if only the observer will acquire the neces- 
sary dexterity and elementary knowledge to enable 
him to study with success. Many who have be- 
come interested in what was at first but rough and 
superficial investigation, have persevered and have 
at length become excellent observers, who have 
added new facts to our knowledge, or have ren- 
dered more accurate information which was already 
possessed." 

It is not intended in this work to give minute 
descriptions of, or to compare one with another the 
microscopes and various apparatus supplied by the 
accomplished opticians in this and other countries, 
the details of which are fully given, and the instru- 
ments and accessories exhaustively described in so 
many works upon the microscope. The aim of 
the present treatise is rather to give thoroughly 
practical directions for the use of the instruments 
and apparatus, so that "research" and manipula- 
tion may go hand in hand. 

For practical work (as distinguished from delicate 
observations with the higher powers) the simpler 
and smaller in other words, the "handier" the 
microscope which is in constant use the better, and 
the lower the power that may safely be used to 



ON INSTRUMENTS AND THEIR USE. 29 

accomplish the work, also, the better. The use of 
the higher powers, and the more delicate accessory 
apparatus of the microscope follows when the pre- 
parations are made, and examination and observa- 
tion commence. As a matter of course, when 
investigations have to be made during the progress 
of the work, powers sufficiently high must be used, 
but let it always be remembered that there is an 
axiom, " all the best microscopical work has been 
done with low powers " which, in reference to the 
aims of this treatise, means all the best and most 
perfect permanent preparations have been made 
with low powers, and are afterwards to be studied 
and examined under the higher powers and con- 
ditions of light, &c., necessary to their elucidation 
and use as valuable aids to scientific instruc- 
tion. For many purposes such as the dissection 
of the larger insects, or the teasing out of the 
coarser tissues, a good " simple " microscope will 
be found extremely useful; but, as a rule, it is 
better to do almost everything under the compound 
microscope, and to patiently acquire the necessary 
skill which enables one to use both ordinary and 
dissecting needles, small brushes, bristles, tubes, 
&c., in the field of the microscope whilst using it ; 
above all things it is important that, if possible, a 
binocular microscope should be used when many 
hours are spent in microscopical work, for not only 
is it natural, and therefore better, to use both eyes, 
but fatigue to the eyes is thus avoided, whilst it is 
certain, in respect of those who work with one eye 
only (unless the eyes are used alternately and often 
changed) that one eye becomes educated at the 
expense of the other, and to such an extent, that 



30 METHODS OF MICROSCOPICAL RESEARCH. 

when the monocular instrument is exchanged for 
the binocular it is often found that the eyes have 
ceased to be a pair, and do not see alike. 

Two eyepieces should be available with the work- 
ing microscope, the ordinary, or A, and a thoroughly 
good C eyepiece, and two objectives. After thirty 
years of constant work the author advises that these 
two powers should be a first rate 1-g- and a J inch. 
These, with the two eyepieces, will give a series of 
four magnifications and prove amply sufficient for 
all ordinary work, and the objectives can be con- 
veniently used with a double nose-piece, except 
when dissecting or other such work is being done 
under the microscope and the nose-piece shall be 
found to interfere with free manipulation. It is 
well to take care that the objectives are as nearly 
as possible of the same length, in order to avoid the 
risk of breaking the slide under the microscope a 
catastrophe very likely to occur either from momen- 
tary forgetfulness of which power may be in use, 
or hurry in changing the objectives. There is no 
difficulty in obtaining a 1^ and J inch objectives so 
arranged as to require but little alteration of the 
rackwork of the microscope to focus them alter- 
nately. Whilst a 1-g- inch objective will be found 
the most useful of all glasses for ordinary work 
and for most purposes, it will often be found neces- 
sary in the course of the work to make examinations 
of its parts and progress by means of the J inch. 
With English microscopes it is advisable, as a rule, 
to use English objectives, but admirable glasses of 
great penetrating power and definition by foreign 
makers can now be obtained at a much less cost 
than those of English manufacture. The object- 



ON INSTRUMENTS AND THEIR USE. 31 

glasses made by Zeiss, Reichart and others, can 
scarcely be surpassed. 

Flatness of field, penetration, definition, and 
entire absence of colour are the essentials of a 
thoroughly good objective, and all objectives not 
possessing these qualities should be rejected. 

For working powers objectives with the lower 
angles of aperture are to be preferred, as it is most 
desirable to secure as much room as possible for 
manipulation between the preparation and the 
objective. For subsequent examination and study, 
however, high power objectives with high angles 
of aperture are invaluable, and oil-immersion objec- 
tives especially so. A thoroughly good fine adjust- 
ment is all-important to a good working microscope. 
The stage of a microscope intended for work should 
be a simple table, either circular or square (pre- 
ferably the former), so that the specimen may be 
rotated and all its parts easily got at ; there should 
be no mechanical arrangements whatever upon it 
(unless these are removable), so that the hands 
may have free scope ; and the stage should be of 
ample size, so that if necessary it may form a rest 
for the hands whilst working. 

For exhaustive observations and delicate investi- 
gations and the study of permanent preparations, 
the elaborate microscopes and beautiful apparatus 
now obtainable are of the highest possible value 
and utility, but for ordinary work they are not only 
quite unnecessary but too complicated to admit of 
satisfactory work being done under them with any 
comfort or convenience, whilst their great costliness 
and delicacy, and their liability to injury from 
chemicals and anything like rough usage, render 
them totally unsuitable as " working " microscopes. 



32 METHODS OF MICROSCOPICAL RESEARCH. 

The following list will prove useful to the student 
who desires to fit up his laboratory, or workroom, 
with the most useful and necessary apparatus and 
requirements for microscopical study, investigations, 
and the making of preparations. 

A good simple microscope, which, however, may 
be dispensed with if the student decides to work 
entirely with the compound microscope as already 
advised. 

A thoroughly good and firm microscope pre- 
ferably a binocular instrument (which can also be 
used as a monocular) with large stage, as already 
described, coarse and fine adjustments, plane and 
convex mirrors, and diaphragm, and having a fitting 
under the stage to receive an achromatic condenser 
of a simple and inexpensive kind, a spot Lens, and 
a Polariscope. 

A Bullseye Condenser. 

Two objectives, 1J and J inch with double nose- 
piece. 

Two Eyepieces, " A" and " C." 

A Camera lucida for drawing objects. 

A Stage micrometer divided into 100th and 
1000th of an inch. 

A good Paraffin Lamp with metal chimney, and 
blue glasses to fit into the chimney to moderate the 
light. 

A retort Stand. 

A Spirit Lamp. 

A small Pestle and morfcar of Glass or agate. 

A best " Cathcart " Microtome arranged for both 
the embedding and Ether freezing processes. 

Two Section Knives and a Plane-iron for cutting 
Sections. 



ON INSTRUMENTS AND THEIR USE. 33 

A set of Scalpels. 

Two pairs of ordinary surgical scissors (small 
and large) and one curved pair. 

Two pairs of forceps (small and large), and a 
specially curved pair for manipulating cover glasses. 

Four " Bulldog " forceps for compressing vessels 
when injecting. 

A pair of Bone forceps. 

Two thick curved needles in handles for lifting 
Sections. 

Needles, small and large, and handles for them 
from which they can be readily removed and 
replaced when it may be necessary to change them. 

Needles with flattened points and cutting edges, 
in handles. 

Two Sewing Machine needles which will be 
found most useful for dissecting and for laying out 
Sections. 

A Bone saw. 

An injecting Syringe, complete with Cannulse 
and Stopcocks. 

A Hypodermic Syringe for delicate injections. 

A writing diamond. 

Two pieces of good cork backed with lead. 

Two small glass dishes for dissecting under 
water. 

Three flasks for boiling. 

A set of Test Tubes and rack. 

Short Tubes, with corks, for storing small 
Sections. 

Half oz., 1 oz. and 2 oz. Bottles, with Corks, for 
storing Sections. 

A Washing bottle. 

Six Dropping bottles. 
3 



34 METHODS OF MICROSCOPICAL RESEARCH. 

Six Dipping Tubes. 

Six Pipettes. 

Three Glass rods for stirring. 

Some small glass tubing. 

Some small india-rubber tubing. 

Six small saucers of white porcelain and six large 
saucers. 

Six Watch glasses. 

Three small Black vulcanite photographic trays. 
These will be found most useful for selecting 
sections, which have been kept in spirit, when 
floated upon water placed in the trays. The black 
background to the white sections renders their 
selection perfectly easy. 

Two small and one large Porcelain Photographic 
Trays, the deeper the better these are for soakiog 
Sections, when stained with Logwood, in Tap 
water. 

Two Glass funnels and a measuring glass for 500 
and one for 10 c.c. 

One gross of 3 in. by 1 in. Glass slips. 

One oz. each of (mixed sizes) thin glass circles 
No. 1 and No. 2. 

Three in. by 1 in. slips with ground out cells. 

Pure Tin cells, -J- in. and f in. in diameter. 

NOTE. Glass slips, thin covers and all tools and apparatus 
required for mounting ; all cements, media and staining fluids, 
&c., can be obtained at Mr. C. Baker's, 244, High Holborn, 
London, W.C. 

LIST OF CHEMICALS REQUIRED. 

Distilled water the purest obtainable. 
Glacial acetic acid. 
Ordinary (best) acetic acid. 



ON INSTRUMENTS AND THEIE USE. 35 

Hydrochloric acid. 

Nitric 

Sulphuric 

Best Methylated Spirit. 

Alcohol, B.P. 

Absolute Alcohol. 

Ammonia B.P. Fort. 

Ammonium Tartrate. 

Alum (powdered). 

Ammonium Chr ornate. 

Ammonium Bichromate. 

Arsenious acid. 

Formic 

Picric 

Osmic ,, 

Acetate of Potass. 

Bichromate of Potass. 

Benzol. 

Borax (powdered). 

Canada Balsam, ordinary and hardened. 

Absolute Phenol (Carbolic acid). 

Carbonate of Potass. 

Ferrocyanide of Potass. 

Carmine (best). 

Celloidin (in Flakes or so-called " shavings "). 

Chloride of Gold (a 15 grain tube). 

Chloroform. 

Chromic acid. 

Clove oil. 

Collodion. 

Ether '720 B.P. 

Gelatine (" Gold label"). 

Glycerin. 

Gold size. 



36 



METHODS OF MICROSCOPICAL RESEARCH. 



Gum arable. 

Hsematoxylin (Crystals). 

Nitrate of Silver. 

Potassium in Cylinders for making Liq. Potass. 

Picrate of Ammonia. 

Sodium Chloride. 

Sulphate of Magnesia. 

Simple Syrup. 

Turpentine. 

Xylol. 

The following tables will be found very useful : 

FRENCH FLUID MEASURES. 

The cubic centimetre, usually represented by "c.c." is the 
unit of the French measurement for liquids. It contains nearly 
seventeen minims of water; in reality, it contains 16-896 
minims. The weight of this quantity of water is one gramme. 

Hence it will be seen that the cubic centimetre and the 
gramme bear to each other the same relation as our drachm 
for solids and the drachm for fluids, or as the minim and the 
grain. The following table will prove to be sufficiently 
accurate for microscopical purposes : 

Cubic 



centimetres. 

1 = 

2 = 

3 = 

4 = 

5 = 

6 = 

7 = 

Q _ 

9 - 

10 = 

20 = 

30 = 

40 = 

50 = 
60 
70 
80 
90 
100 



17 minims (as near as possible). 
34 
51 

or 1 
1 



68 
85 
102 
119 
136 
153 
170 
340 
510 
680 
850 
1020 
1190 
1360 
1530 
1700 



drachm 8 minims. 
25 



,, -L ,, 4HJ 

, 1 42 




, 1 59 




, 2 drachms 16 




, 2 33 




, 2 50 




, 5 40 




, 1 ounce drachm 30 minims. 


, 1 


3 drachms 20 




, 1 


6 


10 




, 2 ou 


ces 1 







, 2 


3 


50 




, 2 


6 


40 




, 3 


1 


30 




, 3 


4 


20 





ON INSTRUMENTS AND THEIE USE. 37 

THE CONVEESION OF FEENCH INTO ENGLISH 

WEIGHT. 

Although a gramme is equal to 15-4346 grains, the decimal 
is one which can never be used in microscopy ; hence in the 
following table it is assumed to be 15f grains, which is the 
nearest approach that can be made to practical accuracy : 
Grammes. 

1 = 15f grains. 

2 - 30* 

3 = 46i ,, 

4 = 6 If ,, or 1 drachm If grain. 

5 = 77 ,,,,1 17 grains. 

6 = 92f 1 32f 

7 = 107J 1 47* 

8 = 1231 2 drachms 3 

9 138f 2 18f 

10 = 154 2 34 

11 = 169f 2 49f 

12 = 184f 3 4* 

13 = 2001 3 20i 

14 = 215f 3 35f 

15 = 231 3 51 

16 = 246f 4 6| 

17 = 261 4 21 

18 = 277i 4 371 

19 = 292f 4 52f 

20 = 308 ,,5 8 
30 = 462 7 42 
40 = 616 ,,10 16 
50 - 770 ,,12* 50 
60 - 924 ,,15 24 
70 = 1078 ,,17 58 
80 = 1232 ,,20 32 
90 = 1386 ,,23 6 

100 = 1540 ,,25 40 



38 



CHAPTER III. 



The Preparation of Animal Tissues. 

THE majority of animal tissues require hardening 
to a greater or less extent, whilst some, on the 
contrary, must be softened before sections can be 
cut from them. The various processes for embed- 
ding and infiltrating organs and tissues have, how- 
ever, recently been brought to such perfection that 
not only can much of the hardening once found 
necessary be dispensed with, much time saved and 
possible injury avoided, but and this is a very 
important consideration the risk of distortions and 
the obliteration of important features and properties 
in the tissues, which it is all important to their 
study should be carefully preserved, are reduced 
to a minimum or altogether avoided by the less 
heroic and more delicate processes now in vogue. 

Hardening agents, in respect of the effects pro- 
duced by them upon tissues, &c., form two divisions: 

(1) those (e.g., alcohol and nitric, and picric acids, 
&c.), which do not interfere with the subsequent 
processes for staining the tissues and sections; 

(2) those (e.^.j osmic and chromic acids, &c.), 
which do more or less affect the delicacy of the 
staining and the action of staining re-agents. 



THE PEEPAEATION OF ANIMAL TISSUES. 39 

In hardening tissues it is all important to secure 
that the specimens shall be thoroughly penetrated 
by the liquid in which they are placed, and to this 
end it is necessary to immerse them in an abund- 
ance of the liquid say into 100 times their bulk, 
or even more and to divide them into sufficiently 
small pieces to ensure this result, regard, of course, 
being had to the attainment of sections of sufficient 
size to show the structure and features of the 
various organs this latter consideration, in these 
days of large sections, being obviously a matter of 
the greatest importance. 

The tissues must be as fresh as possible. Parts 
which have to be treated by the gold or silver 
processes must be immersed within half an hour 
after death, or they will cease to be susceptible to 
the action of re-agents. The time of year also must 
be a matter of consideration, summer and hot 
weather being, of course, less favourable to the pre- 
servation of animal matter than the cold of winter. 
No tissue can be placed too early in the preservative 
medium ; all useless matter, such as the contents of 
the stomach or intestines, must be carefully washed 
away with f per cent, salt solution. As soon as 
a fluid has become " fouled " by the tissues placed 
in it, it should be changed ; as a rule, frequent 
changes are advisable. 

All tissues hardening in chromic acid solutions 
must be examined daily, after the first few days, 
to prevent them from becoming brittle and thus 
spoiled. Different tissues placed in the same 
medium should be separately examined and tested. 
Some may have become sufficiently hardened and 
require immediate removal, whilst a more prolonged 



40 METHODS OF MICROSCOPICAL RESEARCH. 

immersion may be necessary for others. No tissue 
should be allowed to remain in chromium fluids 
until it is quite hard, if, as is commonly the case, 
it is necessary to complete the hardening in alcohol 
to which it should be removed when it has become 
tough, though still retaining its elasticity. When 
transferring a tissue to alcohol to complete its 
hardening, all trace of the medium in which it has 
already been immersed must be removed by pro- 
longed soaking in cold water, which should be 
frequently changed during twelve or twenty-four 
hours, or until the last washings shall be colourless. 
Tissues thus prepared must not be put at once into 
full strength spirit, but first placed in methylated 
spirit and water ("half and half") for twenty-four 
hours, thence into pure methylated spirit. It is 
most important that the shrinkage of the tissues 
caused by the hardening processes should be 
uniform as well as gradual; it is therefore again 
obvious that the greatest care must be taken to 
use fluids which will penetrate the whole tissue or 
organ, lest the surfaces merely should become 
hardened whilst the interior portions remain soft 
and even become decayed. 

As a rule it is advisable to commence with weaker, 
and to advance by degrees to stronger, solutions in 
order to ensure penetration. A book should be 
kept in which notes can be made of the nature of 
the fluids in which organs and tissues are hardening, 
the dates of changing or substituting fluids, &c. 
Each bottle should bear a letter or number to corres- 
pond with the letter or number registered against 
it in the note book. Tissues and organs may be 
wrapped in pieces of linen, and a label bearing its 



THE PREPARATION OF ANIMAL TISSUES. 41 

number to correspond with the note book attached 
to each parcel. In this way many specimens can 
be hardened together in the same fluid, and much 
space and waste of fluid avoided without any risk 
of confusion or error. No hard-and-fast line can 
be drawn in respect of the time during which the 
various organs and tissues should remain in the 
hardening fluids; careful experiments and notes, 
judgment and experience are necessary in this as 
in all other chemical work. Again, many tissues 
require hardening by different processes according 
to the elements in their structure which it is desired 
to demonstrate. For instance, to display the cor- 
puscles and nerves of the cornea it must be treated 
with gold, whilst in order to show the cell spaces 
the silver process must be resorted to. The special 
methods of hardening many of the various tissues 
and organs will therefore be found included under 
more than one process. 

Alcohol. 

When alcohol is employed as the sole hardening 
agent it should be used strong (i.e., of 95 per cent.), 
indeed it will be found necessary in some cases to 
use absolute alcohol. The specimens are to be 
immersed in an abundance of the spirit, which must 
be frequently changed during the first few days. 
It is advisable in many instances to suspend the 
specimens, either wrapped in linen or by a strong 
thread, in the alcohol. Some tissues will become 
sufficiently hardened in a few days, whilst others, 
and especially large specimens and whole organs, 
will require weeks. The more frequently the 



42 METHODS OP MICROSCOPICAL EESEARCH. 

alcohol is changed at first, the more perfect, as well 
as the more rapid, will be the hardening. 

Nitric Acid 

should be used in an aqueous solution of from 5 per 
cent, to 15 per cent., and the specimens may remain 
in it from fourteen to twenty-one days. When it 
shall prove desirable to complete the hardening of 
a specimen in spirit it may be removed from the 
nitric acid and placed in the spirit after a much less 
prolonged immersion in the acid. Great care must 
be taken to remove from the specimen all trace of 
the acid by prolonged soaking in water, frequently 
changed, before placing it in alcohol. 

Chromic Acid. 

Make a 1 per cent, solution in water and reduce 
its strength to J, , or such other percentage as may 
be required. Specimens must be immersed in large 
quantities of this solution, in which they may remain 
for days or even weeks, the length of time during 
which they should be subjected to its action being 
determined by the size and nature of the specimen. 

Mucous tissues harden rapidly in this medium. 
Nervous tissues, spinal cord and brain require weeks. 
Bone from which the muscles (but not the perios- 
teum) have been removed should be steeped in a very 
weak solution, commencing with a ~ per cent, and 
increasing to -J- per cent, with frequent, changes 
during a period of ten days. It is then to be de- 
calcified by immersion in chromic and nitric fluid 
until a needle can be passed through it. It is then 



THE PREPARATION OP ANIMAL TISSUES. 43 

to be washed free from all trace of the acids, and 
transferred to strong spirit. Teeth may be treated 
in the same manner. 

In all cases a weak solution should be employed 
at first and strengthened each time it is changed. 
The specimens should be examined from time to 
time, and removed when they shall have acquired 
the proper condition. The greatest care must be 
exercised lest they become brittle. When removed 
from the chromic acid solution they should be soaked 
in water, frequently changed, for at least twenty-four 
hours, and then transferred, for keeping, to 95 per 
cent, alcohol. 

MUSCLE. A piece of muscle from an animal 
recently killed is steeped in a small quantity of a 
i per cent, solution for a week. The cleavage of 
its sarcous substance is shown by teasing a small 
piece in glycerine. 

NERVE. Harden a piece of metacarpal nerve of a 
horse, or sciatic nerve of a smaller animal for ten 
days in a i per cent, solution. Stain a bit in 
logwood, then tease it in glycerine thoroughly. 
This shows its connective tissue. 

Chromic Acid and Alcohol. 

Mix one part of a 4 per cent, solution of chromic 
acid with two parts of methylated spirit. This 
should be done when required for use. 

The following tissues may be placed in this, then 
transferred to spirit. The time required must be 
judged according to directions already given: 

The whole of one cornea, to show stratified 
epithelium ; a piece of small intestine, to show: non- 



44 METHODS OF MICEOSCOPICAL RESEARCH. 

striped muscle ; heart and pericardium of a small 
animal ; small arteries and capillaries from the 
brain of a sheep, after scraping away the brain- 
substance ; middle-sized artery such as metacarpal 
of the horse; trachea and lungs. These are gently 
injected and then immersed in the fluid. 

The lips, tongue, salivary glands, tonsils, ceso- 
phagus (distended and tied), stomach (after washing 
away its contents with f per cent, salt solution), 
small and large intestines, liver in half-inch cubes, 
ureter and bladder (distended), ovary, fallopian 
tubes, uterus (distended per vaginam) may be 
placed in the solution. 

Besides the above, which may be taken from 
either a dog, cat, or guinea pig, the following 
should be obtained: The thymus gland of an 
infant; skin of scalp, finger and palm of hand, 
sole of foot from the human subject; also a nail. 
The eye of an ox divided transversely just behind 
the cornea for the ciliary muscle, sclerotic, cornea, 
and iris; also the choroid and retina. Of course 
both halves have to be used. The prostate gland 
and penis of a guinea pig. The cervix uteri of 
a cow. Mammary gland of an animal near the 
full period of gestation. The placenta of a cat, 
or guinea pig. The umbilical cord, which must 
be cut into pieces an inch long, and hardened for 
two days in Miiller's fluid before being placed in 
the present medium. 

All the above tissues must be daily examined 
after the third day, and each transferred to spirit 
after it has become tough. Moreover, the fluid 
should be changed after the first twenty-four 
hours, and at intervals of a few days afterwards. 



THE PREPARATION OF ANIMAL TISSUES. 45 

When many different tissues are in fche same jar, 
the quantity of fluid must be such that the upper 
surface of the tissues extends half way up the entire 
fluid, that is to say, the stratum of tissues and 
stratum of clean fluid over them should be of equal 
depth. 

Bichromate of Potass. 

Make a 2 per cent, solution of bichromate of 
potass, with ordinary water. 

Meso-rectum of Cat. Pin this out on cork, and 
float it cork upwards on the solution for seven 
days. 

Liver. After injecting the portal vein with blue 
gelatine mass, and the hepatic artery with carmine 
gelatine mass, the liver may be hardened in the 
solution to toughness, and, of course, finished in 
alcohol. 

Spinal Cord of Ox. Pieces an inch long may be 
placed in the solution frequently changed for 
from three to five weeks. 

Spinal Cord of Ox, Horse, or Sheep. If pieces 
about an eighth of an inch long be macerated two 
or three days in -J per cent, of the solution, 
the anterior horn of the spinal cord snipped out 
with scissors, and teased in carmine solution, then 
pressed with a cover glass, using Farrant's medium, 
they will show the isolated multipolar nerve cells 
very beautifully. The sympathetic nerve cells of 
the frog can be isolated in the same manner. 

Cornea of cat, rabbit, or guinea-pig should also 
be hardened in a 2 per cent, solution for ten 
days. The lens in 1 per cent, solution for one 
week. 



6 METHODS OF MICEOSCOPICAL RESEARCH. 

Ovaries of the Cow in small pieces should be 
macerated in very dilute solution to isolate the 
large, branched, pigmented cells of the Corpora 
lutea. 

Ammonium Bichromate. 

A 2 per cent, solution made with ordinary 
water may be employed. 

This solution is preferred by many to the potass 
salt solution. It is used in the same way. Columnar 
epithelium may be prepared as permanent speci- 
mens by placing a piece of fresh intestine of dog, 
cat, rabbit, &c., in 1 per cent, solution for two 
days ; then steeping an hour or two in water and 
scraping off the epithelium and staining. The cells 
have to be separated with a needle, and may be 
mounted in Farrant's medium, or in glycerine jelly. 

Chromate of Ammonium. 

A 5 per cent, solution is used. 

If a newt's liver (in small pieces) and pieces of 
the small intestines be placed in the solution for 
forty-eight hours, the liver cells and columnar 
epithelium may be obtained, as in the case above- 
mentioned. The goblet cells of Klein can be 
perfectly preserved in glycerine jelly in this way. 
The mesentery of the newt may be placed in the 
solution at the same time, and taken out after 
twenty-four hours. This shows the non-striated 
muscle fibre beautifully. The isolated gastric 
glands of a small mammal may be obtained in 
the same way by placing pieces of the fresh mucous 
membrane for three days in the solution. The 



THE PREPAEATION OF ANIMAL TISSUES. 47 

testes of the newt should be placed in the solution 
for twenty-four hours, then cut into, and their 
contents squeezed out on to a slide. The sperma- 
tozoa are thus obtained as a permanent preparation. 
Another important use of the above solution has 
been pointed out by Heidenhain. If small pieces of 
kidney be placed in the solution for forty-eight 
hours (the cortex should be chosen), the preparation 
shows the cells of the uriniferous tubules and their 
peculiarities as no other method, probably, will 
show them. 

Miiller's Fluid. 

This is made by dissolving twenty-five grms. of 
potass, bichrom. and ten grms. sodse sulph. in 1000 
c.c. of water. 

Miiller's fluid has great penetrating power. It 
hardens slowly, taking, it may be, from five to seven 
weeks. It is useful as a commencing agent, to 
be followed by another of greater shrinking power, 
such as chromic acid and spirit solution, ordinary 
alcohol, &c. Very even shrinkage may thus be 
obtained. For example, if the fresh nasal septum 
is cut out and placed for two days in Miiller's fluid, 
then for a week in chromic acid and spirit solution, 
afterwards in weak, then in pure methylated spirit, 
the olfactory epithelium will be obtained in excel- 
lent preservation. 

After the above explanation no difficulty will be 

found with the following delicate structures, namely: 

Developing tooth, the adenoid tissue of lymphatic 

gland, spleen, thyroid gland, supra-renal-capsules 

(of the horse, by preference), sympathetic ganglion, 



48 METHODS OF MICROSCOPICAL RESEARCH. 

olfactory epithelium, cochlea, testis, epididymis 
and vas-deferens, ovary, human placenta, &c. 

Muller's Fluid and Spirit. 

Take three parts of Muller's fluid and add to it 
one part of methylated spirit and keep it in a dark 
place. The mixture should be made only as re- 
quired. 

This is especially useful for the central nervous 
system. The removal of the brain and spinal cord 
without injury may here be described. Immediately 
after death the skin is removed, or at least the skin 
over the back and neck. Then separate the neck 
and head from the trunk about the middle of the 
neck. Next clear away the muscles on each side of 
the vertebral spines and clip away every spine as 
close as possible with scissors or bone forceps. 
Next with ordinary forceps grasp the laminaB, which 
cover over the spinal cord, one lamina at a. time, 
and break it outwards by inserting one blade of the 
forceps wiihin the neural canal, the other on the 
upper surface of the lamina. Now advance down 
the spine, breaking the laminse outwards right and 
left till all the cord with its membranes is exposed. 
The head portion is dealt with in like manner by 
breaking off, bit by bit, the top of the skull, insert- 
ing one blade into the interior, of course, after 
clearing away the skin and muscles. 

The spinal cord and membranes of a cat, dog, or 
rabbit should be carefully taken out and suspended 
in a deep, narrow vessel. The fluid should be 
changed at the end of twenty-four hours, then after 
a week. At the end of this time, divide into pieces 



THE PREPAEATION OF ANIMAL TISSUES. 49 

an inch long, and continue the hardening for an- 
other week or two, as may be required. It may be 
replaced by a 2 per cent, solution of bichromate of 
ammonium for two weeks, and the pieces preserved 
in spirit, or by Hamilton's method (chloral hydrate, 
twelve grains, water, one ounce). The cere- 
bellum, cerebrum, and, of course, the medulla 
oblongata may likewise be hardened. In the case 
of the two former, divide into suitable pieces to 
ensure perfect penetration. 

The tendo achilles of the calf, and the metacarpal 
nerve of the horse, each in inch-long pieces ; also 
the posterior half of the eye-ball of a pig (for the 
retina) may be hardened with advantage in this 
fluid. 

Absolute Alcohol. 

This should have a specific gravity of 0*795. It 
hardens in twenty-four hours, with much shrinking. 
It is used for secretory glands, notably the pancreas, 
which must be placed direct in it, or the gland may 
spoil by partial self -digestion. The salivary glands, 
the lachrymal glands, pieces from both ends of the 
stomach for the gastric glands, the pancreas in 
pieces, &c., should be placed in it at once. 

If the lymphatic gland of a horse, ox, or smaller 
quadruped is injected with 2 per cent. Prussian blue 
fluid by Klein's method (introduce a glass pipette 
filled with the fluid into a lacteal near a mesenteric 
gland and blow the solution into it), and then placed 
in absolute alcohol, we have the lymph sinuses well 
injected. The muscle structure of the beetle or 
crab is shown well by placing a beetle or the ampu- 
4 



50 METHODS OF MICROSCOPICAL RESEARCH. 

tated limb of a crab for a week in absolute alcohol. 
A bit of muscle is scooped out, stained and mounted, 
after being teased with needles. Non-striped muscle 
may be demonstrated thus : Kill a small animal 
and wash out a length of the , small intestine with 
salt solution, distend it with absolute alcohol, and 
tie both ends, then suspend it in the alcohol for 
twelve hours. With a pair of blunt-pointed forceps 
now tear off strips from the outer surface, which 
will include the longitudinal muscle structure ; stain 
them and mount in balsam. 

Picric Acid. 

Make a cold saturated aqueous solution. Small 
pieces of tissue harden in this in from twelve to 
forty-eight hours. It is excellent for decalcifying 
foetal bones which may be left in it, and tested from 
time to time with a needle, which ought to be 
pushed easily through them. No time can be 
specified, in some cases perhaps weeks may be re- 
quired. Prepare the varieties of cartilage with this 
solution. The aorta (pieces) of a large animal 
(horse, or ox), also a cornea to show its fibrous 
tissue, the thymus gland of an infant, also a lym- 
phatic gland may all be well prepared by remaining 
in this solution twenty-four, thirty-six, or per- 
haps forty-eight hours. The intracellular plexus 
of fibrils may be well shown, thus : Keep a newt 
in a little water for three, four, or five days without 
changing the water. Its outer layer of cuticular 
epithelium is shed as a cast of the entire animal. 
Place the film in the solution for twenty-four hours, 
then wash in plain water till no colour is given off, 



THE PREPAKATION OP ANIMAL TISSUES. 51 

and preserve in common alcohol till required. A 
small piece of this, stained in picro-carmine and 
mounted in Farrant's medium, shows the above- 
named structure beautifully. The fibres of white 
fibrous tissue may be shown to advantage, thus : 
Tear off fine strips of tendo achilles of an animal 
and place them for twenty-four hours in the solu- 
tion. By " teasing " a little piece in water the 
white fibres held together by cement substance are 
well displayed. 

Osmic Acid. 

This is purchased as a 1 per cent, solution and 
diluted with distilled water to a ^, a J per cent, 
solution, &c., as required. It is a hardening agent 
which also stains fatty matter, and is, therefore, 
useful in blackening the medullary nerve fibres. It 
is very poisonous, very expensive, and soon spoiled 
by exposure to light. It must be kept in a well 
stoppered bottle, which should be of black glass or 
covered with black paper, so that not a particle of 
light shall be admitted. Substances placed in it 
harden in from four to thirty-six hours, and may be 
prepared and mounted in either glycerine, glycerine 
jelly, or Farrant's medium. Very little pieces, not 
larger than half a grain of wheat, are to be used of 
any tissue, and the bottle in which they are placed 
must also be very small ; the short glass tubes in 
which homoeopaths keep their smallest pilules 
answer well. 

With the above cautions and directions we may 
enumerate the tissues which are treated with 
advantage by osmic acid. In a 1 per cent, solution 



52 METHODS OP MICROSCOPICAL RESEARCH. 

immerse costal cartilage of a kitten, puppy, or 
young rabbit, rat or guinea pig, for twelve hours ; 
a piece of the sciatic nerve of the frog for ten 
minutes ; the non-medullated nerve running in the 
wall of the splenic vein of an ox, also a piece of 
human placenta. A 1 per cent, solution may be 
injected into a testis with a hypodermic syringe, 
and the whole organ may then be placed in alcohol. 
The following are best treated with a -J- per cent, 
solution, namely : liver cells of a dog or small 
animal (the liver is cut across and the cut surface 
scraped) are placed in the solution for an hour. 
Areolar tissue is demonstrated by injecting a -J- 
per cent, solution into the groin of a puppy or 
kitten immediately after death. A bulla is thus 
formed, which must be snipped out with scissors, 
spread on a slide stained with logwood and then 
covered. A per cent, solution may be forcibly 
injected into the anterior horn of a fresh spinal 
cord. The part is cut out, macerated for two days 
in dilute alcohol, and the multipolar cells isolated 
by " teasing," after staining with picro-carmine. 
A J per cent, solution (the strength most 
frequently employed) may be used for showing 
mucous tissue ; thus : inject the axilla, or groin, 
of a very young embryo, as described above. 
Pieces the size of a pea of the following may be 
hardened in a J per cent, solution : Submaxillary 
gland, fresh pancreas, pieces from each end of 
the stomach, small intestines, supra-renal cap- 
sules of the horse, human skin. Place a piece of 
the anterior horn of a fresh spinal cord for ten 
days in a ifeth per cent, solution, then, after washing 
away the greyish deposit, place it in an equal 



THE PREPARATION OF ANIMAL TISSUES. 53 

quantity of glycerine and water for a fortnight ; 
stain with weak magenta solution, " tease " in 
glycerine, and cover (Stirling). 

Miscellaneous Preparations. 
Salt Solution. 

This is made as a f per cent, solution, that 
is, 75 grm. in 100 c.c. of water. It is used for 
washing away foreign matter from organs before 
immersing them in other fluids, and for examining 
fresh tissues. In so using it, a very small drop is 
placed on a slide, and in it is immersed a minute 
piece of the structure about to be examined. 
"Tease" this out with needles; then put over it 
a cover glass. 

The following are to be studied : Fresh columnar 
epithelium from the small intestines ; ciliated epithe- 
lium scraped from the roof of the frog's mouth; 
ciliary motion may be seen in the yellowish coloured 
gills of the common salt water mussel. Ligamen- 
tum nuchse of ox ; subcutaneous connective tissue ; 
adipose tissue ; red marrow from a long bone ; 
striped muscle (best seen in sartorius of the frog) ; 
nerve fibre, sciatic of the frog ; fresh pia mater ; 
fresh spleen (the ox spleen by preference) ; thymus 
gland ; kidney ; Gasserian ganglion ; placenta ; 
decidua, &c., may all be so treated. 

Glycerine. 

Margarine crystals may be obtained by steeping 
morsels of fat for twenty-four hours in glycerine. 
These post-mortem products appear as delicate 
needles. 



54 METHODS OP MICROSCOPICAL RESEARCH. 

Water. 

Squamous epithelium may be stained in magenta 
solution and examined in water. To obtain it, 
scrape the inside of the cheek with a blunt knife. 
The surface of the cheek may be also scraped and 
the scraping examined in water and afterwards 
irrigated with a 5 per cent, solution of liquor 
potassae. 

Dilute Alcohol. 

Mix two parts of water with one of rectified 
spirit. This is a useful dissociating solution recom- 
mended by Ranvier. 

Olfactory, ciliated and transitional epithelium, 
may be prepared as permanent preparations thus : 
Place a small piece of fresh trachea of a small quad- 
ruped for ciliated a piece of fresh bladder for 
transitional the head of a frog, with the nostrils 
slit up, for olfactory epithelium, in the solution for 
two days. The parts are then scraped and the 
scrapings stained and mounted in glycerine, or 
glycerine jelly. Stirling " fixes " the ciliated 
epithelium by placing the scraping in a 1 per cent, 
solution of osmic acid. Non-striped muscle may 
be obtained by taking out the bladder of a fresh- 
killed frog and distending it with the solution, then 
placing it for twenty-four hours in the solution. 
After brushing away the mucous membrane with a 
camel-hair brush, stain the bladder in picro- 
carmine, and mount a piece in Farrant's medium. 
Isolated heart muscle fibres of the frog's and mam- 
malian heart can be obtained by the same process. 



THE PREPARATION OF ANIMAL TISSUES. 55 

Purkinje's fibres can be obtained by snipping out 
bits of them (seen as fine transparent lines on the 
inner parts of the walls of the ventricles of the 
heart of oxen and sheep), and placing them in the 
solution for two days. The villi of human placenta 
can be isolated by soaking a piece of placenta two 
days in the fluid. 

Chromic Acid and Nitric Acid Solution. 

This is made by adding 1 c.c. of strong nitric acid 
to every 100 c.c. of the J per cent, solution of 
chromic acid. It is used chiefly as a decalcifying 
agent. 

Tooth, intervertebral disc with subjacent bone, 
articular cartilage with subjacent bone^ costal 
cartilage of an old person, may each be placed in a 
large quantity of the fluid, which must be frequently 
changed, until the bony part is softened, which may 
be known by the needle test. The parts are washed 
free of the acids by frequent changes of water, then 
placed in weak, afterwards in strong, alcohol for 
preservation. 

Permanent preparations of elastic tissue may be 
made thus : Soak half-inch cubes of ligamentum 
nuchae of ox or horse in this fluid for a week; wash 
free of acids and preserve in spirit, and make trans- 
verse and longitudinal sections in the usual way. 
(Stirling). 

Silver Nitrate. 

All animal tissues which are to be subjected to 
nitrate of silver and chloride of gold processes, 
must be perfectly fresh and carefully washed in 



56 METHODS OF MICROSCOPICAL RESEARCH. 

distilled water before being treated by either of 
these re-agents. 

Make a 1 per cent, solution of nitrate of silver 
in distilled water, keep it in a bottle carefully 
covered all over with black paper. This solution 
is to be further diluted to a -J, a ^, or a J 
per cent, as required. Nitrate of silver is used 
to intensify and demonstrate the outlines and 
features of endothelium and cartilage cells. The 
cement substance between the cells absorbs the 
solution all not absorbed must be carefully washed 
away with distilled water, and the tissue exposed 
to direct sunlight. Not the slightest taint of the 
unabsorbed silver solution must be allowed to re- 
main upon the surface of the tissue, the staining 
of which would be spoiled by the black oxide 
granules of decomposed nitrate of silver. The 
tissues must be treated within a few minutes after 
death. 

Omentum of rabbit. Kill a rabbit by bleeding. 
Remove the omentum and wash it in distilled water; 
place it in a J per cent, solution ten minutes ; wash 
in ordinary water thoroughly ; place the whole in 
a saucer containing water, and expose to diffuse 
daylight till slightly brown. Small pieces may now 
be stained with logwood and mounted in glycerine 
or glycerine jelly, and the logwood stain may be 
dispensed with if desired. 

Septum Cysternce Lymphaticce magnce of a frog. 
Kill a frog and immediately open the abdomen. 
Gently push on one side the stomach, bowels, &c., 
and pour distilled water on the part behind the 
stomach, when a delicate membrane floats up. 
Now pour a -J per cent, solution, drop by drop, 
over this till it becomes milky, and treat as above. 



THE PREPARATION OF ANIMAL TISSUES. 57 

Lungs of a kitten, or puppy. Distend these with 
a \ per cent, solution immediately after death, then 
ligature the trachea, and sink them in alcohol till 
required. 

Tendon. Tendons and their sbeaths are covered 
by endothelium. Pinch the tail end off a recently 
killed mouse, and wash the fine tendon fibres thus 
drawn out in distilled water, and treat with a ^ per 
cent, solution. Take another set of these fibres, 
pencil off their endothelium with a camel-hair brush 
dipped in distilled water, then silver. This shows 
the cell spaces in tendon. 

Cornea. The cell spaces of cornea may be shown 
by scraping away the epithelium from the anterior 
surface of the cornea of a pithed frog, then applying 
a 1 per cent, solution till greyish white. The 
cornea must be snipped at its edges, after being 
silvered, to make it lie flat on the slip. A rat's 
cornea may be likewise treated and stained with 
logwood. 

Blood Vessels. Kill a small quadruped by bleed- 
ing. Syringe out the blood vessels with distilled 
water; then inject a % per cent, solution. Use the 
spleen, mesentery, and intestines. The spleen must 
be hardened in alcohol, and sections cut, exposed 
to light, after staining with logwood, or not, as 
desired. This shows the endothelium of the venous 
sinuses. The intestines must be cleared of their 
contents by distilled water ; exposed to light in a 
saucer containing water ; a piece of small or large 
intestine is snipped out, laid on a slide with its 
mucous membrane upwards, which is gently scraped 
away, the muscular and serous coats remain, and 
are to be mounted in balsam. 



58 METHODS OF MICROSCOPICAL RESEARCH. 

Membranous Connective Tissue. The omenta of 
an adult cat and rabbit, also of a young rabbit, are 
to be treated like the omentum of rabbit as already 
described, to show the membranous connective 
tissue. The omentum of the young rabbit shows 
developing fat cells and blood vessels. Some of the 
above should be stained with logwood. 

Adenoid Tissue. "With a hypodermic syringe in- 
ject a fresh lymphatic gland with a J per cent, 
solution, and after placing it for twenty-four hours 
in alcohol, make sections. These are stained with 
logwood, and exposed to light till brownish. 

Cement Substance of Non-striped Muscle. Washout 
a small length of intestine of a rabbit with distilled 
water ; then fill it with a per cent, solution, and 
tie both ends ; then place it in a J per cent, solution 
for a quarter of an hour. Wash away all the silver, 
and cover over with water in a saucer. Thin 
Iamina3 of the outer muscular fibres are to be 
stripped off with broad nibbed forceps. Mount 
some stained with logwood in balsam ; others un- 
stained, as desired. These also show the lymphatics. 

Sciatic Nerve of a Frog. To show Ranvier's- 
crosses, kill a frog, and dissect out the sciatic nerve, 
wash it in distilled water, then place a piece a line 
in length in a J per cent, solution for five minutes. 
Wash it now thoroughly in water and tease it care- 
fully in glycerine, cover, and expose to light till 
brownish. Mount a small length of an entire thick- 
ness of an intercostal nerve of a rat or mouse, to 
show its endothelial covering. Of course it is not 
to be teased, and it must be very carefully washed 
or the endothelium will be destroyed. 

Lymphatics of the Diaphragm. Expose the 



THE PREPABATION OF ANIMAL TISSUES. 59 

posterior (ventral) surface of a rabbit or guinea- 
pig, immediately after death by bleeding, thus : 
Ligature the gullet and the posterior vena cava, 
then remove all the abdominal viscera. Tie up by 
the hind legs, then with a brush dipped in distilled 
water brush away all the epithelium covering the 
centrum tendinuin of the diaphragm. "Wash silver 
solution over it, and treat as already explained. 
This silvers the endothelia lining the lymphatics. 

Chloride of Gold. 

This salt is sold in small tubes containing 15 
grains. A 2 per cent, solution is to be made with 
very pure distilled water, and kept in a black 
bottle, like the silver solution. The operator must 
also provide himself with formic acid and two or 
three fresh lemons. 

Development of Capillaries. Snip off the tail of a 
half-grown tadpole, place it in a watch glass, and 
squeeze the juice from a fresh lemon over it, and 
let it remain immersed for five or ten minutes ; 
wash it in distilled water to remove all the juice ; 
then steep it for half-an-hour in a 1 per cent, solu- 
tion. Wash away the surplus gold, then place in 
a mixture of one part of formic acid and three 
parts of water for twenty-four hours in a cool place 
and away from the light. The gold chloride will 
then be reduced. 

The following are to be treated in exactly the 
same manner : 

A whole Cornea. That of a pig, dog, or cat, to 
show the nerves of the cornea, also the cornea 
corpuscles. 



60 METHODS OF MICROSCOPICAL RESEARCH. 

A Piece of Skin. This should be taken from the 
snout of a pig or a mole, or both ; also the soft 
part of a duck's bill. Sections show the nerves of 
skin. 

Striped Muscle. The recti muscles of a rabbit's 
eye are to be taken, a strip cut off lengthwise. 
These show nerve terminations in striped muscle. 

Muscle ivith its Tendon. Take a piece of the 
diaphragm of a rabbit ; let the piece be part of the 
centrum tendinum with its attached muscle. Sec- 
tions made in the long axis of the muscle fibres 
show the terminations of muscle in tendon; that 
is to say, the connecting links are shown. 

Tendon from the Tail of a Mouse. After treating 
a few leashes as above, snip off a piece a line in 
length; tease in glycerine and cover, or simply 
press the cover glass upon it till it flattens out. 
This preparation shows the relations of the cells 
and fibres in tendon. 

Tail of Rat. After treating as above, with the 
exception that it must remain an hour in the gold 
solution, decalcify, by placing in chromic and nitric 
solution, then make transverse sections and stain 
with logwood. 

Nerve Ganglia. Treat the heart of a frog, in 
small pieces, as above ; dissect out the nerves and 
ganglia. These are also found lying along the 
course of the abdominal aorta of the frog, and may 
be taken from there if preferred. The nerve 
ganglia of the bladder and ureter of a small mammal 
may likewise be thus demonstrated. 



THE PREPARATION OF ANIMAL TISSUES. 61 

The Injection of Blood Vessels. 

Blood and lymphatic vessels are more satisfac- 
torily demonstrated when filled. The former are 
better injected with a fluid which becomes solid in 
ordinary temperatures. The vessels must be fully 
distended at the time and this distention must 
remain. Gelatine forms the foundation of injection 
masses for the above purposes, because it can be 
liquefied at blood heat and it solidifies in a little 
lower temperature; then, again, it is capable of 
being easily cut, and does not become brittle, but 
remains tough and resistant, though sufficiently 
soft. Injections of different colours are employed 
in order that the arteries may be distinguished 
from the veins, and these again from other 
channels, such as bile ducts, lymphatic vessels, 
capillaries, &c. 

The distribution of the capillary vessels, indeed, 
in many cases cannot be satisfactorily studied or 
demonstrated without injection or other special 
modes of preparation. Injections of single organs, 
or parts carefully dissected from the body of an 
animal, all the larger arteries and vessels being 
carefully tied, can be made ; whilst the entire 
blood system of mammalian animals, including 
man, of birds and reptiles, can be injected from an 
arterial trunk, so that the finest capillary vessels 
shall be perfectly filled. 

Red Mass. 

This is made of gelatine coloured with carmine. 
In making it the greatest care must be exercised in 



62 METHODS OF MICROSCOPICAL BESEARCK. 

order to ensure a neutral or at least a slightly acid 
mass ; because if it should have any alkaline re- 
action, it will diffuse through the vessels, stain the 
adjacent tissues, and thus render the preparation 
altogether worthless. It is preferable that the 
mass should be slightly acid, but if too much so, 
granulation of the carmine will ensue, and the fluid 
will not be driven into the arterioles far less into 
the capillaries. Parts injected with a carmine and 
gelatine mass must be immersed in equal parts of 
water and methylated spirit, with 1 per cent, of 
acid added thereto. 

DR. CARTER'S CARMINE MASS. 

Take of Carmine, 60 grains. 

Strong Ammonia, 120 minims. 

Glacial Acetic Acid, 86 minims. 

Solution of French Gelatine (gold label), 1 part to 6 

parts of Water, 2 ounces. 
Water, 1^ ounce. 

Dissolve the carmine in the ammonia and water, 
filter if necessary. With this mix 1J ounce of the 
hot solution of gelatine. Mix the acetic acid with 
the remaining half ounce of gelatine, and drop this 
mixture, very slowly, into the carmine and gelatine 
solution, with constant stirring. 

DR. STIRLING'S CARMINE MASS. 

Take of Carmine, 60 grains. 

Strong Ammonia, 60 minims. 
Glacial Acetic Acid, 80 minims. 
Gelatine, 1 ounce. 
Water, q.s. 

Soak the gelatine in water for several hours ; 
pour off the water, which is not absorbed, when the 



THE PEEPAEATION OF ANIMAL TISSUES. 63 

gelatine is completely saturated and swollen, and 
melt it in a water bath. Strain, whilst hot, through 
flannel and make up the solution to 2 ounces. 
Place the carmine in a mortar, add to it the 
ammonia and 2 ounces of water, and leave it for 
twelve hours. Filter, and add the acetic acid, drop 
by drop, stirring all the while, until the ammonia 
is completely neutralised. As the odour of the 
ammonia becomes faint, the acid must be added 
very cautiously. As long as there is free ammonia, 
the fluid is dull red, but it becomes of a brilliant 
colour as soon as the ammonia is neutralised. Now 
mix the two solutions at a temperature of 40 C. 

DR. G-. SIMS WOODHEAD'S MASS. 

Take of Carmine, 4 parts by weight. 

Liq. Ammonia, 8 parts by measure. 

Gelatine (Cox or Coignet's), 10 parts by weight. 

Distilled Water, 100 parts by measure. 

Put the carmine in a mortar, and pour on the 
ammonia, when an almost black paste will be 
formed if the carmine is pure ; pour in the water, 
and set the solution aside to be filtered. Place the 
gelatine in a narrow glass jar, and add sufficient 
distilled water to cover it, and allow it to stand 
until the gelatine is thoroughly softened. Warm 
the carmine solution in a pan of water, kept at 
nearly boiling point over a Bunsen gas burner, and 
add the gelatine; stir thoroughly, add a 10 per cent. 
solution of acetic acid, drop by drop, until the 
alkalinity of the ammonia is neutralised, and the 
fluid shall be even slightly acid. The point at 
which this takes place will be recognised by the 
pungent odour of the ammonia becoming fainter and 



64 METHODS OF MICROSCOPICAL RESEARCH. 

fainter, and that of the acid substituted, whilst the 
fluid loses its bright carmine, transparent colour, 
and turns a dull brownish-red. 

BLUE INJECTION MASS. 

Take of Soluble Prussian Blue, 4 drachms. 
Gelatine, 4 ounces. 
Distilled Water, 20 ounces. 

Thoroughly mix the blue powder in a mortar 
with half the water. Treat the gelatine as directed 
in the case of the red mass, and add the blue solu- 
tion very gradually, and with constant stirring, to 
the liquefied gelatine. Filter through fine flannel. 
The greatest care must be exercised that no trace 
of alkali shall enter into the composition of this 
mass all the vessels used in making it should be 
carefully washed with acidulated water, and the 
purest distilled water must be used in its prepara- 
tion. Its colour is liable to fade and sections 
injected with it, especially when mounted in 
balsam, lose their colour after a time. It is better, 
therefore, to mount all specimens injected with 
Prussian blue in a slightly acid medium glycerine 
jelly prepared with boracic acid pure glycerine 
with a small proportion of acetic acid, or Farrant's 
medium are the best preservatives for these injec- 
tions. 

BLUE INJECTION MASS. 

(Robin's modification of Dr. Beetle's Prussian Blue) is made 
as follows : 
Take (a) Sulphocyanide of Potassium, saturated solution 

90 c.c. 

Glycerine, 50 c.c. 

(b) Liquid perchloride of Iron at 30, 3 c.c. 
Glycerine, 50 c.c. 



THE PREPARATION OF ANIMAL TISSUES. 65 

Mix slowly, and combine the mixture with three 
parts of a glycerine-gelatine injection mass made 
as follows (Robin's formula) : 

Dissolve, in a water bath, 50 grammes of French 
gelatine (gold label) in 300 grammes of distilled 
water in which some arsenious acid has been dis- 
solved ; add 150 grammes of glycerine and a few 
drops of carbolic acid. 

SCHEELE'S G-REEN INJECTION MASS (Robin's formula). 

Take of Arseniate of Potash (saturated solution), 80 c.c. 
Glycerine, 50 c.c. 

and of 

Sulphate of Copper (saturated solution), 40 c.c. 
Glycerine, 50 c.c. 

Mix and combine with three parts of the gly- 
cerine gelatine injection mass as in the preceding 
formula. 

Aqueous Injection Fluids. 

CARMINE. (Emery's formula.) 

To a 10 per cent, ammoniacal solution of carmine 
add acetic acid, with constant stirring, until the 
colour of the solution becomes brilliant red. After 
the precipitation of the carmine, pour off the super- 
natant fluid and inject the clear solution. Place 
the injected specimens in strong alcohol to fix the 
carmine. 

CARMINE. (Gerlach's formula.) 

Carmine, 77 grains. 
Distilled water, 70 grains. 
Liquor ammoniae, 8 minims. 

Dissolve the carmine in the ammonia and water, 
and leave the solution exposed to the air until all 
5 



66 METHODS OF MICROSCOPICAL RESEARCH. 

the ammonia has evaporated. Dissolve a drachm 
and a half of best gelatine in a drachm and three- 
quarters of water ; add a few drops of glacial acetic 
acid. This fluid is to be injected warm. 

ACID CARMINE FLUID. (Dr. Scale's formula.) 

Carmine, 5 grains. 

Glycerine, with 8 to 10 minims of acetic acid, ^ ounce. 

Glycerine, 1 ounce. 

Alcohol, 2 drachms. 

Distilled water, 6 drachms. 

Ammonia, a few drops. 

Mix the carmine with a few drops of water, and 
when thoroughly incorporated, add 5 minims of 
liquor ammonia. Add about half an ounce of the 
glycerine, and shake the mixture well in a bottle. 
Next, pour in very gradually the acid glycerine, 
frequently shaking the bottle during admixture. 
Test the fluid from time to time with blue litmus 
paper, and if not of a very decidedly acid reaction, 
add a few drops more acid to the remainder of the 
glycerine and mix as before. Lastly, add the 
alcohol and water, very gradually, shaking the 
bottle thoroughly after the addition of each succes- 
sive portion, until the whole is well mixed. 

PRUSSIAN BLUE FLUID. (Dr. Beale's formula.) 

Wood-naphtha, or pyro-acetic spirit, 1| drachm. 

Glycerine, 2 ounces. 

Alcohol, 1 ounce. 

Ferrocyanide of Potassium, 12 grains. 

Tincture of Perchloride of Iron, 1 drachm. 

Distilled Water, 3 ounces. 

Dissolve the ferrocyanide of potassium in 1 
ounce of the glycerine, mix the tincture of iron 



THE PREPAKATION OF ANIMAL TISSUES. 67 

with the other ounce. Put this into a bottle. Add 
the iron mixture, drop by drop, to the ferrocyanide 
solution, shaking all the time. Mix the naphtha 
with the alcohol, add the water very gradually, and 
thoroughly shake the whole. 

PRUSSIAN BLUE ACIDULATED GLYCERINE FLUID. 

(Dr. Beetle's formula.) 

Price's Glycerine, 2 fluid ounces. 
Tinct. of Sesquichloride of Iron, 10 minims. 
Ferrocyanide of Potassium, 3 grains. 
Strong Hydrochloric acid, 3 minims. 
Distilled Water, 1 ounce. 

Dissolve the ferrocyanide in one half of the 
glycerine, and the sesquichloride of iron in the other, 
add the latter drop by drop to the former. Finally 
add the water and the acid, and to the whole add 
2 drachms of alcohol. 

NEUTRAL GLYCERINE PRUSSIAN BLUE FLUID. 
(Dr. Beale's formula.) 

Glycerine, 1 ounce. 

Alcohol, 1 ounce. 

Ferrocyanide of Potassium, 12 grains. 

Tincture of Perchloride of Iron, 1 drachm. 

Distilled Water, 4 ounces. 

Dissolve the ferrocyanide in one ounce of the 
water and glycerine, add the tincture of iron to 
another ounce. Mix these solutions very gradually, 
in a bottle, and shake thoroughly. The iron is to 
be added to the ferrocyanide solution. Lastly, the 
alcohol and the remaining water are to be gradually 
added, with constant shaking of the bottle. 

Specimens injected with this fluid should be 
preserved, and mounted, in acidulated glycerine. 



68 METHODS OF MICROSCOPICAL RESEARCH. 
NEUTRAL GLYCERINE TURNBULL'S BLUE FLUID. 

(Richardson's formula.) 

Ferridcyanide of Potassium, 10 grains. 
Sulphate of Iron, 5 grains. 
Distilled Water, 1 ounce. 
Price's Glycerine, 2 ounces. 
Alcohol, 1 drachm. 

Proceed as in the last formula. Mix the sulphate 
of iron in 1 ounce of the glycerine, and the ferrid- 
cyanide in a small quantity of water, and mix this 
with the other ounce of glycerine. Gradually mix 
these two solutions., with constant shaking, and add 
the alcohol. 

Turnbull's blue is not so liable to fade as 
Prussian blue. 



69 



CHAPTER IV. 



On Injecting Blood Vessels, &c. 

IT has been generally, though erroneously, con- 
sidered that a perfect injection of an animal cannot 
be secured until the rigor mortis has passed away; 
it is, however, indubitable that the most successful 
injections have been made immediately after death. 

Dr. Beale 1 says: " I have found that most perfect 
injections may be made before the muscular rigidity 
begins, that is, within a few minutes after the death 
of the animal. Most of my fine injections have 
been made less than five minutes after death, and 
in the case of very young animals, so complete has 
been the injection of the capillary vessels, that 
where the capillary has not been fully developed, 
the injection has filled the pervious portion, and 
has penetrated to the very spot where the tube was 
commencing to be formed." This the author can 
entirely corroborate. 

The ordinary process for injecting blood vessels 
is with the syringe ; as this is a very delicate opera- 



" How to Work with the Microscope," fifth ed., 1880, p. 
114. 



70 METHODS OF MICROSCOPICAL RESEARCH. 

tion requiring much dexterity of manipulation, 
considerable practice, some experience and great 
care, the student must not be discouraged by 
disappointments and failures, but persevere until 
success is attained. It is advisable to practise with 
cold aqueous fluid media, and this operation being 
successfully accomplished, to go on to injections 
with the warm gelatine "mass." It will be found 
best, also, to commence by injecting the organs of 
animals, such as the kidney, liver, lung, large and 
small intestines, &c., of the cat, the sheep, or the 
pig, the eye of an ox (from the artery), and by 
complete injections (from the aorta), of small 
animals, e.g., rat, guinea-pig, rabbit and frog, 
before attempting to inject a whole animal. 

The kidney should be singly injected with 
carmine mass through the artery, and a double 
injection should also be made with carmine from 
the artery, and with blue from the vein. 

The liver in like manner may be injected from 
the portal and hepatic veins and from the bile ducts 
(thus ligature the common bile duct, remove 
the fundus of the gall bladder, remove as much bile 
as gentle pressure will force out, affix the cannula, 
and inject). 

In injecting isolated organs removed from the 
body of an animal, the greatest care must be taken 
to tie all divided vessels, and to use no more than 
the necessary pressure upon the piston of the syringe. 
The slower and more gradual the flow of the fluid, 
the more perfect will be the injection. 

All animals, or " parts " to be injected with a 
gelatine mass must be placed in warm water of a 
temperature from 40 to 45 C. during the process. 



ON INJECTING BLOOD VESSELS, ETC. 71 

To INJECT AN ANIMAL WITH A GELATINE " MASS " 
BY THE SYEINGE. 

Arrange so that they may be ready to hand 
the necessary instruments and apparatus, viz., the 
syringe, thoroughly cleaned and tested for work, 
with its stopcocks and cannulae, scalpels of several 
sizes, a large and a small pair of scissors, dissecting 
forceps, " bull-dog " forceps (for clamping leaking 
vessels), a curved needle threaded with silk or 
thread for tying vessels, a wash-bottle of a capacity 
of from half a pint to a pint. The injection mass is 
to be melted by standing the vessel containing it in 
hot water, and it is to be kept fluid in a water-bath 
of a temperature from 40 to 45 C. 

Chloroform the animal (say a rabbit) which is to 
be injected, and make an L-shaped incision into the 
thorax so as to expose the heart and aorta. This 
is done by cutting upwards along the middle line of 
the sternum nearly as far as the root of the neck, 
then making a second incision at right angles to 
this, to the rabbit's left. A triangular flap is thus 
made and the heart, enclosed in the pericardium, 
exposed. Having cut through the pericardium, 
seize the apex of the heart with a pair of forceps 
and snip it off. The right and left ventricles are 
thus opened, and the animal instantly bleeds to 
death. The opening in the right ventricle, leading 
to the pulmonary artery, has a crescentic, slit-like 
appearance, whilst the opening in the left ventricle, 
leading to the aorta, is circular. Insert the cannula 
into the aorta and tie it in securely with a silk 
ligature. Wash all blood from the cavity of the 
thorax in order to ensure the cleanliness of the 



72 METHODS OF MICROSCOPICAL RESEARCH. 

water-bath. Now place the rabbit into a warm 
water-bath of a temperature of from 40 to 45 C., 
and let it remain for from 10 to 15 minutes, i.e., 
until thorougly warmed. The entire abdomen may 
be carefully opened along the middle line to allow 
the warm water to surround the internal organs, 
and to ensure the equable flow of the injected fluid 
into all parts of the body. 

When the animal is thoroughly warm the 
syringe, which should also have lain for some time 
in warm water is to be carefully filled with the 
injecting fluid, in which its nozzle is to be entirely 
immersed, whilst the piston is worked several times 
to ensure the expulsion of all air. It is then placed 
in the stopcock, which should, however, first be 
filled with fluid from the syringe to prevent the 
introduction of any air, and which has already been 
inserted into the neck of the cannula to receive the 
nozzle of the syringe, and which, stopcock, is to be 
turned off each time it may be necessary to refill 
the syringe ; now with a gentle equable pressure 
the fluid is to be injected into the animal until the 
entire system of blood vessels shall be filled, but not 
distended. The operator from time to time examines 
the head, opens the eyes and parts the lips of the 
animal to watch the progress of the work. If the 
fluid is flowing properly, the mouth will almost 
immediately exhibit signs of the colour with which 
the injection is being made, and which will first be 
seen in the gums of the lower incisor teeth. When 
the eyeballs show the finest lines of colour traversing 
them, the capillaries may be considered to be filled 
and the injection complete and perfect. 

The animal is now to be put into cold water in a 



ON INJECTING BLOOD VESSELS, ETC. 73 

large vessel, and placed under a tap which is to be 
allowed to drip slowly upon it for two hours; it 
may then be dissected and the organs placed in 
70 per cent, methylated spirit for twenty-four 
hours and then transferred to strong methylated 
spirit in which they are to be preserved. 

If it is desired to inflate the lungs, remove them 
entire; very carefully tie all vessels and let them 
remain in the warm water. Melt together and keep 
fluid in a warm water-bath, two parts of cacao 
butter and one part of lard, suspend the lungs in 
the water-bath, insert a funnel into the trachea, 
and pour the butter and lard gradually into the 
lungs until they are fully inflated but no more 
great care must be exercised not to over-distend 
them. Now place them in cold water and after- 
wards into spirit as already described. 

Dr. Fearnley has devised an elaborate and most 
effective modification of Ludwig's constant pressure 
apparatus for making injections, which he fully 
describes in his " Practical Histology," 1 and which 
ensures such constancy, accuracy and delicacy in 
the pressure upon, and flow of, the injecting fluid, 
that there can be no comparison between its almost 
automatic action and that of the syringe worked by 
a piston. 

This apparatus consists of a bath, having a 
shallow part to receive the animal to be injected, 
and a deeper part for the vessel containing the 
injection fluid to stand in. A large (40 ounce) 
Wolff's bottle, with three necks, is fitted with three 



1 " A Course of Elementary Practical Histology," by 
William Fearnley, p. 70. 



74 



METHODS OF MICROSCOPICAL RESEARCH. 



perforated india-rubber stoppers. A glass tube 
reaching nearly to the bottom of the bottle passes 
through the central stopper; each of the other 
stoppers is fitted with a glass tube projecting but 
little beyond the stopper into the bottle, but pro- 




jecting sufficiently above the stopper to admit of 
a piece of india-rubber tubing, of the diameter used 
for infant's feeding bottles, being fixed upon it. 
A basin, or still better, a water-bottle, of a 



ON INJECTING BLOOD VESSELS, ETC. 75 

capacity of a litre or more, a mercurial manometer, 
a Higginson's syringe, a spirit lamp, or a Bunsen's 
gas burner, some glass and brass cannulae, and 
some india-rubber tubing, two clamps (one for 
the pressure tube, the other for the delivery tube), 
will also be required. 

To make an injection of an animal with a gelatine 
mass, proceed as follows : 

Fill the water-bath with water of a temperature 
of from 40 to 45 C. Keep it at this temperature 
by placing a spirit lamp or a Bunsen gas burner 
under the bath. Melt the gelatine mass and pour 
it, through flannel, into the two-stoppered Wolffs 
bottle. Place this bottle in the place provided for 
it in the bath the bottle should be weighted to 
prevent its floating as the fluid is withdrawn from 
it. Arrange the manometer, pressure bottle, syringe 
and basin (or water-bottle), as shown in illustration. 
Now chloroform the animal to be injected, and pro- 
ceed as already described ; affix the cannula, tie it 
securely into the aorta, and after washing away all 
blood from the thorax, &c., place the animal in the 
water-bath. The animal must be submerged in the 
water, but not too deeply. Clamp the delivery tube 
of the bottle containing the injection fluid and, with 
the syringe, pump into the pressure-bottle from 
the basin or water-bottle sufficient water to raise 
the mercury in the manometer one inch; remove 
the clamp from the delivery tube, allow the air to 
escape from it, and immediately connect it with the 
canuula. The tube must remain submerged whilst 
the connection is being made. Now keep up an 
equable pressure by means of the syringe, and when 
the mercury in the manometer shall have risen to 



76 METHODS OF MICROSCOPICAL RESEARCH. 

from 4 to 5 inches, it will be found that the animal 
is completely injected. The head must be examined 
from time to time, as already described, so that the 
rising colour of the lips, gums, tongue and eyes may 
be watched. The animal is now removed to cold 
water, and treated as directed above. 

This method of applying pressure is wonderfully 
delicate and equable; it will be found that whilst 
the mercury in the manometer can be raised almost 
imperceptibly, one entire compression of the barrel 
of the syringe raises it 1 inch. 



77 



CHAPTER V. 



On Staining Fluids, and Staining. 

THE value of the various staining processes 
consists in the perfection with which the differen- 
tiation of the tissues is accomplished, and their 
features and structure brought out. In the stain- 
ing of tissues it is all important that the nuclei of 
the cells shall be deeply stained, whilst the cellular 
structure is clearly defined. 

Staining is an art, requiring much experience 
and great delicacy of manipulation. It is one thing 
to colour a section or a tissue, quite another to 
stain, and especially to double-stain and differen- 
tiate it. In double and triple staining the great 
desideratum is to secure a thoroughly good nuclear 
stain as the ground colour, and to supplement this 
with a delicate tint, or tints, which shall prove a 
good contrast to the first stain and thoroughly 
differentiate the tissue without diffuseness. 

For staining the nuclei of the cells, for beauty 
and permanence, no staining fluids have yet been 
discovered to surpass haematoxylin, carmine and 
picro-carmine. The brilliancy of carmine has its 
advantages in many cases, whilst the delicacy, the 



78 METHODS OP MICROSCOPICAL RESEARCH. 

beautiful gradations of tints, from the deepest violet 
to the most delicate blue, which by careful treat- 
ment are produced by a good logwood stain, give a 
sense of relief to the eye during prolonged observa- 
tions which no other tints, perhaps, afford. No 
more beautiful stainings have been produced than 
those obtained by a prolonged immersion (say for 
twelve hours) in a logwood solution, made according 
to the first formula given below, and diluted to 
a light violet colour, which after the sections 
so stained have lain in tap water for twenty- 
four hours will result in a most beautiful blue 
ground colour with the nuclei of a darker blue. 
Let them then be placed in spirit for half an 
hour, and then in a stain made from 2 parts of a 
saturated alcoholic solution of rubin and 1 part of a 
saturated alcoholic solution of yellow eosin diluted 
with alcohol, until it becomes of a delicate pink 
shade, and in which the sections may be kept until 
they are " cleared " and mounted. The sections 
thus treated will be found to be distinctly triple- 
stained in blue, crimson and orange colour without 
diffuseness and with perfect differentiation, and if 
mounted in " xylol balsam' 9 they will prove quite 
permanent. 

Carmine and picro-carmine also are invaluable as 
nuclear stains, whilst for many tissues, and especially 
for most vegetable tissues, they are preferable to 
logwood, and, when mounted in a suitable medium, 
sections stained with them prove equally beautiful 
and permanent preparations. 

A carefully selected list of formulas for the most 
important and reliable staining re-agents is here 
appended, the proper media in which sections 



ON STAINING FLUIDS, AND STAINING. 79 

stained by them should be mounted being given 
with each formula. 

HJIMATOXYLIN STAINING FLUIDS. 

1. Haematoxylin (Crystals), 1 gramme. 
Alum powdered, 10 grammes. 
Distilled Water, 100 c.c. 
Alcohol, 5 c.c. 
A small piece of Camphor. 

This is a splendid nuclear stain. 

It does not become active until from two to three 
months after it is made. It improves by keeping, 
and remains perfectly good for two or three years. 
It must be filtered each time it is used, and may be 
used over and over again. Its full strength is too 
strong for use ; lor rapid staining, i.e., in from five 
to ten minutes, add to it an equal volume of distilled 
water. For slow staining, dilute it until its colour 
becomes a light violet, and leave the section in the 
stain thus diluted for six to twelve hours. Over- 
stained sections can be decolourised to any desired 
tint in distilled water 5 parts, acetic acid 1 part. 

After staining and, if necessary, decolourising 
the sections, place them in tap water for twenty- 
four hours, and they will be found of a beautiful 
blue tint. Mount in xylol balsam. 

2. EHRLICH'S FOEMULA. 

(a) Haematoxylin, 1 to 2 grammes. 
Absolute Alcohol, 100 c.c.m. 

(6) Glycerine, 100 c.c.m. 

Distilled Water, 100 c.c.m. 

Glacial Acetic Acid, 5 c.c.m. 

Alum Sulph. (powdered), to saturation. 

Mix the two solutions. 



80 METHODS OF MICROSCOPICAL RESEARCH. 

Allow it to stand for about six weeks, occasionally 
shake it, when it will have obtained its greatest 
depth of colour, then filter it once for all. It 
will then stain in about half an hour or less and 
does not overstain. But better results are obtained 
by adding a few drops to an ounce of distilled water 
and leaving the sections therein for from three to 
four hours. It keeps well, and as it gets older, 
stains more rapidly. If its action becomes too 
rapid or diffuse, add more acetic acid. It may be 
used over and over again. 

Mount in xylol balsam. 

3. WEIGERT'S FORMULA. 

(a) Hsematoxylin (Crystals), 1 gramme. 
Absolute Alcohol, 10 c.c. 
Distilled Water, 100 c.c. 

(b) Ferridcyanide of Potassium, 2| grammes. 
Borax (powdered), 2 grammes. 
Distilled Water, 100 c.c. 

Place the sections in absolute alcohol for one 
hour then in solution (a) until they become quite 
black then place them in distilled water for a few 
minutes. Decolourise in solution (6) until they are 
of a dark brown. 

This does not keep good and active beyond a few 
months. Filter each solution every time it is used, 
and it may be used several times. Mount in xylol 
balsam. This is a very fine nuclear stain, and brain, 
spinal cord, nerves, &c., are superbly stained by it. 
It is also a splendid stain for micro-photographic 
purposes, as the beautiful shades of brown which 
carefully stained sections exhibit give very fine 
photographic results. Any animal or vegetable 



ON STAINING FLUIDS, AND STAINING. 81 

sections, of which it is desired to produce lantern 
slides or micro -photographs, may be stained by this 
process. Mount in xylol balsam. 

4. KLEINENBERG'S FORMULA. 

(a) Prepare a saturated solution of chloride of 
calcium in alcohol (of 70 per cent.), add a small 
quantity of powdered alum, filter, mix 1 part of this 
with 8 parts of 70 per cent, alcohol. 

(&) Make a saturated solution of haematoxylin 
crystals in absolute alcohol. When using this for 
staining purposes add to a small quantity of 
solution (a), in a watch glass or saucer, sufficient of 
(b) to give it the desired tint. 

This is a very fine nuclear stain, and permanent, 
but the stain itself will not keep very long ; all 
sections or tissues to be stained in it must be 
thoroughly soaked in distilled water so that no 
trace of acid may remain in them. 

Slow staining in a very dilute solution gives the 
best results. Mount in xylol balsam. 

5. MITCHELL'S FORMULA. 

Take of finely ground Logwood, gii. 
"Alum Potash, 5ix. 
Glycerine, fl. %iv. 
Distilled Water, quant, sun . 

Moisten the logwood with water until it is quite 
damp. Place it in a large funnel with filter paper 
and then pass ordinary water through it untilj the 
liquid runs away almost colourless. When all the 
water has drained from it, place the logwood on a 
large dish to dry. 

Dissolve the alum in 8 ounces of distilled water, 
6 



82 METHODS OP MICROSCOPICAL RESEARCH. 

place the logwood in a vessel of sufficient size and 
pour the alum solution upon it, allow it to remain 
for forty-eight hours, stir it occasionally, strain it 
off; add the glycerine and filter. Add 5 per cent, 
of absolute alcohol to it, and keep it in a thoroughly 
well stoppered bottle. For rapid staining this fluid 
may be used full strength, but better results will be 
obtained from prolonged immersion, say for twelve 
hours, in a weak solution. This stain will give a 
delicate violet tint to the sections with beautiful 
clearness and differentiation. Mount in xylol 
balsam. 

Carmine Staining Fluids. 

1. AMMONIA CARMINE. (Dr. Heale's Formula.) 

Carmine, 10 grains. 

Liquor Ammonia for t., B.P., | drachm. 

Price's Glycerine, 2 ounces. 

Distilled Water, 2 ounces. 

Alcohol, | ounce. 

Dissolve the carmine in the ammonia by means of 
heat. Boil for some seconds and allow it to cool. 
Let the ammonia evaporate for an hour or more, 
and when the odour is nearly gone, add the 
glycerine, water and alcohol, and filter. When, 
from keeping, the carmine is precipitated, add one 
or two drops of ammonia. It may be used full 
strength or diluted with alcohol, which will increase 
its penetrative power. This is a fine, nuclear stain. 

Mount in xylol balsam. 

2. RANVIER'S FORMULA. 

Distilled Water, 100 parts. 
Ammonia, 1 part. 
Carmine, 1 part. 



ON STAINING FLUIDS, AND STAINING. 83 

Rub up the carmine in a mortar with a small 
quantity of the water, add the ammonia, and when 
the carmine is dissolved, add the remainder of the 
water. If there is an excess of ammonia, use heat 
until the carmine begins to be precipitated. 

Mount in xylol balsam. 

3. HUXLEY AND MAETIN'S FORMULA. 

Carmine, 2 grammes. 

Liquor Ammoniae/or., B.P., 4 c.c. 

Distilled Water, 48 c.c. 

Dissolve the carmine in the ammonia and water. 
Place it in a bottle without a stopper until all odour 
from the ammonia has nearly passed away. Keep 
it in a thoroughly good stoppered bottle. When 
used it should be diluted with from fifteen to twenty 
times its bulk of water. 

Mount in xylol balsam. 

4. BORAX CAEMINE FLUID. 

(Foster and Balfour's Formula.) 

Carmine, 3 grammes. 
Borax, 4 grammes. 
Distilled Water, 97 grammes. 

Mix, apply heat with constant stirring until the 
liquid nearly boils, set it aside to cool and when 
cold, add 

Alcohol (Eectif.), 70 c.c. 

Distilled Water, 30 c.c. 

Shake it up thoroughly, let it stand for thirty-six 
to forty-eight hours, and filter it. 
Mount in xylol balsam. 
Specimens stained in these carmine fluids may be 



84 METHODS OF MICROSCOPICAL EESEAECH. 

rendered more brilliant, and any diffuseness re- 
moved by immersion in 

Hydrochloric Acid, 1 per cent, in Distilled Water, 1 part. 
Alcohol (Rectif.), 2 parts. 

PICEO-CAEMINE STAINING FLUIDS. (Gage's Formula.) 

1. Take of carmine and of picric acid equal 
parts by weight. Dissolve the picric acid in 100 
times its weight of distilled water. Heat may be 
used if necessary. Dissolve the carmine in 50 
times its weight of liquor ammonia fort., B.P. 

Mix the two solutions, filter through thick filter- 
paper, evaporate the filtered liquid to dryness, 
dissolve the filtrate in 100 times its weight of 
distilled water. Filter until a perfectly clear solu- 
tion results. Then add to each 100 c.c. of the 
solution, 

25 c.c. of Glycerine (Price's). 
10 c.c. of Absolute Alcohol. 

This will keep perfectly well, but should be 
filtered at intervals of a few months. 

2. WEIGEET'S FOEMULA. 

Soak 2 grammes of carmine for twenty-four 
hours in 4 grammes of ammonia. Place it under 
a glass shade to prevent evaporation, add 200 
grammes of a very strong solution of picric acid, 
and replace it under the glass shade for a second 
period of twenty-four hours. Then add, very 
gradually, acetic acid until precipitation com- 
mences. Again put it aside for twenty-four hours. 
Filter, then add ammonia in small quantities drop 
by drop at intervals of twenty-four hours until the 



ON STAINING FLUIDS, AND STAINING. 85 

solution becomes clear. If this fluid when used 
overstains yellow, add acetic acid, if red, add 
ammonia. 

3. PiCRO-LiTHiuM CARMINE. 



Dissolve 2J grammes of carmine in 100 c.c. of a 
saturated solution of lithium carbonate, add 2 to 
3 c.c. of a saturated solution of picric acid. Filter. 

All preparations stained with picro-carmine may 
be mounted in xylol balsam, but Farrant's medium 
is preferable. 

PICRO-CARMINE AND EOSIN. (Lang's Formula.) 

Mix 50 parts of a 1 per cent, picro-carmine 
solution with 50 parts of a 2 per cent, aqueous 
solution of eosin ; after prolonged immersion for 
from twelve hours to forty-eight, or even seventy- 
two hours wash out the picrin in repeated 
changes of alcohol, until the eosin ceases to colour 
the spirit. 

This is a very fine double stain. 

Mount in xylol balsam. 

STJLPH-INDIGOTATE OF SODA. 

Make a saturated solution of sulph-indigotate of 
soda in distilled water, and to each ounce thereof 
add from 3 to 6 minims of rectified alcohol, filter, 
and keep in a closely stoppered bottle. It is better 
to make small quantities of this fluid as required, 
as it does not keep very long. This stain is suit- 
able only for fibrous tissues skin, scalp, hair, and 
for epitheliomatous growths. 

Mount in xylol balsam. 



86 METHODS OP MICROSCOPICAL RESEARCH. 

Cochineal Staining Fluid. 

ALCOHOLIC COCHINEAL. (Mayer's Formula.) 
Macerate cochineal powder in 70 per cent, alcohol 
for some days, in the proportion of 10 c.c. alcohol 
to 1 gramme of the cochineal. 
Stir frequently and filter. 

Saturate the specimens before staining them with 
70 per cent, alcohol, and use alcohol of that strength 
for all the processes connected with the use of this 
stain, and for diluting it. After staining the speci- 
mens, wash them in repeated changes of the alcohol 
until they cease to tint it. 

ALUM COCHINEAL. (Czoker's Formula.) 

Pulverise in a mortar, cochineal 7 grammes, and 
dried alum 7 grammes; add 700 c.c. of distilled 
water, and boil down to 400 c.c. When cool add 
1 per cent, of carbolic acid crystals ; filter several 
times. It will remain fit for use for about six 
months, when it must again be filtered, and a small 
additional quantity of carbolic acid added to it. 

Use it full strength, it does not over stain how- 
ever long the specimens are left in it. 

The specimens must be immersed in it for at least 
two hours, and may require to remain immersed 
from three to six hours. 

It acts as a double stain, colouring the nuclei 
violet and the tissues red. 

ANILIN BLUE BLACK. 

Dissolve 1 decigramme in 4 c.c. of distilled water, 
to this add 100 c.c. of rectified alcohol, and keep 
the solution in a well- stoppered bottle. 



ON STAINING FLUIDS, AND STAINING. 87 

Stain the specimens in this fluid much diluted 
with spirit. The stained specimens may be kept, to 
be mounted as required, in spirit to which a small 
quantity of this fluid has been added. 

This is a most valuable stain for brain, spinal 
cord, nerve centres, and the nervous system gener- 
ally, also for bone and cartilage. 

Mount in xylol balsam. 

Anilin Dyes as Staining Fluids. 

These are valuable for many purposes, and have 
many useful properties. They stain rapidly, with 
great clearness and brilliancy, and are especially 
useful as double and triple stains in conjunction 
with haematoxylin or carmine. 

No more beautiful or effective staining process 
has been discovered than that in which the speci- 
mens, having been stained in hsematoxylin fluid 
subjected to the action of " tap " water for twenty- 
four hours, until they have acquired a beautiful 
blue tint, are afterwards stained in a weak solution 
of 2 parts f uchsin and 1 part yellow eosin. 

This process gives a clear and pure triple stain. 

The student will find by experiment many equally 
useful and beautiful combinations of these dyes. 

It has always been affirmed that most of the 
anilin colours are more or less, and indeed, more 
rather than less, fugitive, but the author ventures to 
affirm as the result of his long experience that if 
the necessary precautions are taken there is no 
reason whatever why specimens stained with anilin 
colours should not remain as permanent as any 
others, and he has in his collection preparations so 



88 METHODS OF MICROSCOPICAL RESEARCH. 

stained, made twenty years ago, which are as per- 
fect as on the day they were mounted. 

Since xylol has been introduced anilin stains may 
be used with even greater confidence, as that pre- 
paration tends rather to preserve than to destroy 
the anilin colours, which are undoubtedly adversely 
affected by benzol. Xylol, therefore, should be used 
to clear the specimens before mounting them, and 
Canada balsam, which has been thoroughly hardened 
and re- dissolved in xylol, should be used as the 
mounting medium. If the specimens, having been 
cleared in a mixture of equal parts of xylol and 
absolute phenol, are mounted in " xylol balsam, 5 ' 
brilliant clearness will result, and the preparations 
will prove absolutely permanent. 

The author has found it most convenient to make 
saturated solutions in alcohol of the anilin colours 
given in the appended list, and to stain the specimens 
slowly in weak solutions, either alcoholic, made by 
adding a few drops of the saturated solution to 
alcohol, or aqueous, made by adding a small 
quantity of the stain to distilled water. The 
advantage of using alcoholic solutions is that when 
the dilute stain has been found to give the desired 
tint it may be preserved for use over and over 
again. It must be filtered each time, and as its 
colour becomes lighter, must be restored to its 
original tint by the addition of a few drops of the 
" stock " solution. 

The following will be found the most generally 
useful of the anilin colours and sufficient for all 
purposes. 

Anilin Blue. 

Nicholson's Blue. 



ON STAINING FLUIDS, AND STAINING. 89 

Soluble Blue. 

Bismarck Brown. 

Chrysoidin. 

Eosin (Red). 

Eosin (Yellow). 

Fuchsin. 

Gentian Violet. 

Acid Green. 

Iodine Green. 

Magenta. 

Methyl Green. 

Methyl Violet. 

Orange. 

Rubin. 

Saffranin. 

Spiller's Purple. 

Vesuvin. 

Bismarck Brown. Make of this (and of all the 
above) a saturated solution in alcohol (all these 
solutions should be filtered at intervals of a few 
weeks). Add a few drops to a small staining saucer 
full of spirit, and place the specimens in it for a few 
minutes. Remove them to alcohol, clear in xylol 
and phenol and mount in xylol balsam. 

If it is desired to mount the specimens in Farrant's 
medium, or glycerine, wash them first in alcohol, then 
in distilled water. Bismarck Brown does not over- 
stain, nor does it wash out readily either in water or 
alcohol. It is suitable for bacteria and micrococci, 
and forms a very good double stain in conjunction 
with other colours : its brown tints render it a good 
stain for preparations intended for micro-photo- 
graphy. 

Eosin. Both red and yellow eosin are most use- 



90 METHODS OF MICROSCOPICAL RESEARCH. 

fill stains either singly or in conjunction with other 
staining fluids, and especially with hgematoxylin ; 
whilst, as before stated, yellow eosin combined with 
fuchsin gives, with hsematoxylin as the ground 
stain, a precise and beautiful triple stain. 

Eosin has great staining and especially penetrat- 
ing powers, and specimens may remain in a weak 
alcoholic solution for an indefinite time without fear 
of their becoming over-stained. No trace of acid 
must remain in any medium in which eosin stainings 
are mounted ; but preparations mounted in xylol 
balsam are practically permanent. 

Epithelium is beautifully stained by eosin and 
the blood of the frog or triton double stained with 
hsematoxylin and eosin forms a superb preparation. 

Iodine Green. Add to distilled water, in a 
staining saucer, sufficient of the saturated solution 
in alcohol to give it a fairly deep tint. Place the 
specimens in distilled water for a short time before 
staining them. Iodine green stains very rapidly, 
and in many cases instantaneously. Wash them in 
water, dehydrate in absolute alcohol, clear in xylol 
and phenol, and mount in xylol balsam. Stained 
specimens may remain in a weak alcoholic solution 
of this dye indefinitely and be mounted as required. 

Methyl Green. This is an intense nuclear 
stain, and Dr. Curschmann states that it has "a 
peculiar affinity for amyloid substance, colouring it 
an intense violet; whilst surrounding tissues that 
have not undergone degeneration are stained green 
or bluish green." 

Methyl Violet is also a valuable stain for 
tissues which have undergone amyloid degenera- 
tion : the amyloid substance is stained red, and the 
unaffected parts violet. 



ON STAINING FLUIDS, AND STAINING. 91 

Orange and Rubin. These combined, in 
about the proportion of 1 part of the former to 2 
parts of the latter, give, in conjunction with haema- 
toxylin as the ground stain, a beautiful triple 
stain. 

Saffranin is a good nuclear stain, imparting a 
beautiful rose tint to the tissues, and staining the 
nuclei a very deep red. 

Spiller's Purple is also a good nuclear stain, 
and is a very restful colour for the eyes. 

Vesuvin, in conjunction with anilin blue, is very 
effective as a stain for bacilli and bacteria. 

Specimens stained with anilin dyes must not be 
cleared in oil of cloves or cedarwood oil, but exclu- 
sively in xylol and absolute phenol in exactly equal 
proportions. This medium clears almost instantane- 
ously and perfectly, having also a preservative 
rather than a destructive effect upon the anilin 
colours of which it increases the brilliancy ; whilst, 
moreover, the xylol balsam, in which they should 
be mounted, exercises a preservative effect upon 
the preparations, and which it renders practically 
permanent. 

(Haematoxylin crystals, and the anilin dyes, are 
supplied of the finest quality by Dr. G. Griibler, of 
Leipzig, and can be obtained from his agent, Mr. C. 
Baker, 244, High Holborn, London, W.C.) 

The Staining of Bacilli and Bacteria. 

To stain bacillus tuberculosis : 
FORMULA No. 1. 

1. 1^ Fuchsin, 1 gramme. 

(a) Absolute Alcohol, 10 c.c. 
Carbolic Acid, 5 grammes. 

(b) Distilled Water, 100 c.c. 



92 METHODS OF MICROSCOPICAL RESEARCH. 

Mix the two solutions. (This remains active for 
about two months.) 

2. Anilin (or Methylene) Blue, 2 grammes. 
Absolute Alcohol, 15 c.c. 
Distilled Water, 85 c.c. 

(This will keep a long time.) 

Spread a thin layer of sputum on a thin glass 
cover, dry, and fix it over a spirit lamp or Bun sen's 
gas burner until it commences to give off vapour; 
then float the cover, sputum downwards, upon the 
surface of a small quantity of fluid (1) in a staining 
saucer ; leave it for fifteen minutes. 

In the case of sections, immerse them in the fluid 
for twenty minutes. 

Now decolourise in 1 part of sulphuric acid to 
7 parts of distilled water, until all colour has dis- 
appeared. Wash away all trace of the acid in 
distilled water, when a slight tinge of colour will 
become perceptible. Float the cover upon, or 
immerse the sections for a few minutes in, solution 
2. Wash in distilled water, place in absolute 
alcohol for three or four minutes. Let the cover 
become perfectly dry, and mount in xylol balsam. 
In the case of sections dehydrate them in absolute 
alcohol, clear them in xylol and phenol, and mount 
them in xylol balsam. (The xylol in which the 
balsam is dissolved will clear the sputum on the 
cover.) 

2. DR. HENEAGE GIBBES' FORMULA. 

1^ Magenta Crystals, 2 grammes. 
Pure Anilin, 3 grammes. 
Absolute Alcohol, 20 c.c. 
Distilled Water, 20 c.c. 



ON STAINING FLUIDS, AND STAINING. 93 

Float a thin glass cover upon which sputum has 
been spread or immerse sections, as in the former 
process, in this fluid, for twenty minutes. 

Decolorise in a 33 per cent, solution of nitric acid 
in distilled water, wash free from acid and float the 
cover upon, or immerse the sections for a few 
minutes in, a saturated solution of chrysoidin in 
distilled water ; when they are stained brown, wash 
them in distilled water, place them in absolute 
alcohol for a short time. Clear the sections in 
xylol and phenol, and mount in xylol balsam. 

If a small crystal of thymol is dissolved in absolute 
alcohol and added to each of the solutions in the 
above two formulas it will tend to their preservation. 
Each of these solutions should be filtered into the 
staining saucers when used. 

EHELICH'S PROCESS. 

Make a saturated solution of phenylamin in 
distilled water. This is done by prolonged shaking 
up the water with the floating anilin and filtering ; 
add to the filtered mixture a saturated solution of 
fuchsin in absolute alcohol until a brilliant red 
colour is obtained. Now, as in the former processes, 
spread a thin layer of sputum upon a thin glass 
cover and dry, and fix it over a spirit lamp or 
Bunsen's gas burner. Float the cover upon the 
fuchsin solution for from fifteen to twenty minutes, 
wash it in distilled water and float it upon a 33 per 
cent, solution of nitric acid in distilled water for a 
few seconds, when it will be found quite colourless. 
Now float the cover for a few minutes, on solution 
No. 2, of formula No. 1. Wash in distilled water, 
dehydrate in absolute alcohol, mount in xylol 
balsam. 



94 METHODS OF MICROSCOPICAL RESEARCH. 

WEIGERT'S PROCESS for Schizomycetes. 
Bacilli, Bacteria and Micrococci. 

5t A 2 per cent, solution of Gentian Violet in Distilled 
Water, 12 c.c., and a Saturated Aqueous Solution 
of Anilin oil (filtered) 100 c.c. 

Stain the sections in this. 

Make a Solution of Vesuvin, 1 gramme. 
Eectified Alcohol, sp. gr. 830, 10 c.c. 
Distilled Water, 100 c.c. 

Stain in this for fifteen minutes. 
Clear in xylol and phenol. 
Mount in xylol balsam. 

REV. GORDON THOMPSON'S PROCESS. 

The special feature of this valuable process con- 
sists in fixing the bacilli, bacteria or micrococci, 
after staining them with the ground colour, by 
subjecting them to the action of a solution of gold 
made by dissolving 15 grains of gold chloride in 4 
ounces of distilled water. This solution is to be 
poured upon the cover, on which the bacilli have 
been spread and allowed to remain upon it for two 
or three seconds and then poured off again, the 
cover is then to be washed in distilled water. The 
gold appears to form a tube round the bacteria and 
so encased, they appear to be rendered absolutely 
permanent ; preparations, in the author's possession 
mounted by this process twelve years ago, remaining 
as brilliant in colour as the day they were made. 

For the ground stains 

Methyl violet, fuchsin and acid green are used, 
the covers being floated upon the solution, washed 
in distilled water, then treated with the gold 
solution, and again washed in distilled water. 



ON STAINING FLUIDS, AND STAINING. 95 

For the second stains use 

Acid green, Bismarck brown or chrysoidin ; wash 
in distilled water. 

Mount in pure glycerine jelly containing no trace 
of acid. 

The best contrasts of colour will be found to be 

Fuchsin followed by acid green. 

Fuchsin followed by anilin (or soluble) blue. 

Bismarck brown followed by anilin (or soluble) 
blue. 

Methyl violet followed by acid green. 

Clearing Media. 

Specimens which are to be mounted in Canada 
balsam or any other resinous media must be pene- 
trated by a " clearing " agent, which shall render 
them perfectly transparent and remove from them 
all traces of the alcohol in which they have been 
dehydrated or kept. It is now generally admitted 
that hardened balsam, which has been re-dissolved 
in xylol is the best and most reliable preparation 
of that most useful, valuable and permanent mount- 
ing medium. 

It is obvious that a clearing agent which consists 
in great measure of the solvent of the balsam must 
be peculiarly adapted to precede the balsam, and it 
may at once be said that the best, the cleanest in 
its application, and the easiest to use, consists of a 
mixture of exactly equal parts of xylol and absolute 
phenol. The manner of its use is simplicity itself, 
whilst its penetrative and " clearing " actions are 
practically instantaneous. Much has been said and 
written about elaborate processes for running the 



96 METHODS OF MICROSCOPICAL RESEARCH. 

clearing agent under the sections, letting them sink 
through alcohol into the clearing agent placed at 
the bottom of a test tube or saucer, with alcohol 
above it, raising the sections with " lifters," &c., 
but this complicated process tends to the destruc- 
tion of the sections, which cannot be too little 
manipulated. Now, in xylol and phenol we have 
a medium which requires no such treatment, all 
that is necessary being to place the fluid in a saucer 
to the depth of about a quarter of an inch. The 
saucer should be a large one, so that there shall be 
no difficulty in removing the sections, or any 
necessity for dragging them up the side of the 
saucer. Place the sections for five minutes in 
absolute alcohol, transfer them, with a thick curved 
needle which has a blunt point, to the surface of the 
clearing fluid. The alcohol is almost instantly 
removed and the sections sink to the bottom. Then 
take a perfectly clean cover, and with the curved 
needle float a section carefully on to the cover and 
spread it perfectly flat, drain 'the excess of the fluid 
on to a piece of blotting paper, by holding the cover 
in forceps and placing its edge upon the blotting 
paper, then lay the cover section upwards upon the 
blotting paper to remove the clearing fluid from its 
under surface, and before the clearing fluid evaporates 
apply xylol balsam to the section. The balsam 
being miscible with the clearing fluid, there is 
nothing antagonistic or greasy to remove, which is 
the case when oil or turpentine, or both, are em- 
ployed as clearing agents. 

This medium is the best that can be used for 
clearing sections cut from specimens which have 
been embedded in celloidin, upon which it has no 
solvent effect. 



ON STAINING FLUIDS, AND STAINING. 97 

When, however, it is not desired to mount with 
the section its surrounding and supporting celloidin, 
Turpentine will be found the best solvent of that 
material as well as of paraffin when that has been 
used as the embedding agent. 

Turpentine should not be used for specimens or 
sections which have been hardened or preserved in 
alcohol or methylated spirit, as it has great shrink- 
ing power. It will be found very useful for entomo- 
logical preparations, and especially for insects with 
very hard integuments ; these must be left to soak 
in turpentine until they are cleared, but small and 
delicate insects can be better cleared in xylol and 
phenol. Turpentine is also a good clearing agent 
for vegetable specimens of a woody nature. All 
specimens or sections cleared by turpentine should 
be mounted in Canada balsam, hardened and re- 
dissolved in turpentine, and carefully filtered. 

Oil of Cloves has some advantages over all other 
clearing agents (xylol and phenol excepted), and is, 
perhaps, the most universally used. It combines 
well with both alcohol and balsam and does not 
shrink the specimens as turpentine does ; but 
"clearing" with this oil involves its removal by 
placing the sections in turpentine after they have 
been cleared, because if the clove oil remains in the 
specimens or sections, although it is miscible with 
the balsam, it prevents the balsam from hardening 
and so renders the preparations unsafe. 

Sections cut from specimens which have been 
embedded in celloidin and which it is desired to 
mount so embedded must not be cleared in oil of 
cloves, as it dissolves the celloidin at once ; it is 
therefore a good clearing agent when it is desired to 
7 



98 METHODS OF MICROSCOPICAL EESEARCH. 

remove celloidin. It must be remembered that oil 
of cloves renders specimens which have been 
allowed to remain in it very brittle. 

Oil of Bergamot is a valuable, though costly 
agent ; it clears the celloidin embedded specimens 
and does not affect anilin stains to any great extent. 
It is fairly rapid in its action and clears well. 

Cedar Wood Oil. This oil is not expensive, 
but is very slow in its action, it also has the valuable 
properties of clearing perfectly, however slowly, 
and of not injuriously affecting anilin dye's, or of 
shrinking specimens which have been hardened or 
preserved in alcohol. It is very useful for clearing 
vegetable sections, though extremely slow in doing 
so, and celloidin sections may be cleared in it, but it 
takes several hours to render them transparent. 

Mounting Media and Cements. 

Many media in which specimens may be examined 
and delicate dissections made for examination, are 
not in the true sense of the term preservative and 
therefore serve only a temporary purpose. Many 
solutions again which re-act upon tissues, and 
elucidate their features and composition serve only, 
in like manner, for purposes of temporary examina- 
tion and study. It is intended, in the following list 
of media in which specimens and sections can be 
mounted, to give the formulae and methods of using 
and applying the best preservative media so as to 
secure the permanence and safety of the prepara- 
tions which are made with them. The list will be 
supplemented with formulae for the most useful 
fluids in which to make dissections and to place 



ON STAINING FLUIDS, AND STAINING. 99 

tissues or specimens for temporary observation and 
examination only. 

Canada Balsam. The most universally used 
and useful of all mounting media is the oleo-resin 
obtained from Abies Balsamea and Pimis Canadensis 
and is mostly imported from Quebec. The balsam 
is obtained from the tree by puncturing the blisters, 
or vesicles, which form under the bark of the trunk 
or its branches, and collecting their fluid-contents 
in a bottle. It is at first opaque, but gradually 
clarifies and becomes transparent. It consists of 
24 per cent, of volatile oil, 60 per cent, of resins 
soluble in boiling alcohol, and of 16 per cent, of 
resins soluble in ether, but insoluble in alcohol. 

As it hardens very slowly, it is, for histological 
purposes and for use as a mounting medium, 
generally evaporated to dryness and re-dissolved 
in either benzol or xylol, preferably in the latter. 
These solutions harden rapidly, are perfectly 
transparent, practically permanent and preservative. 
For some exceptional purposes, e.g., for specimens 
which require to be mounted in deep cells, pure, 
undiluted balsam will be found useful. 

In order to prepare Canada Balsam for histo- 
logical purposes it should be gently heated over a 
sand bath and thus slowly evaporated until it be- 
comes quite hard. Great care must be taken that 
the heat does not become too great so that the 
balsam is burned, in which case it becomes of a 
dark brown colour and is consequently spoiled. 
When quite dry and brittle it is to be reduced to a 
coarse powder and re-dissolved in an equal quantity 
of one or other of the solvents above named. For 
delicate entomological and some vegetable prepara- 



100 METHODS OF MICROSCOPICAL EESEAECH. 

tions hardened balsam may be re-dissolved in 
turpentine all these solutions of balsam, whether 
in xylol, benzol or turpentine must be carefully 
filtered for use, and should be kept in wide-mouthed 
capped bottles. 

1. GLYCERINE JELLY. 

1^ Glycerine (Price's), 50 c.c. 

Distilled Water, 42 c.c. 
1 Boracic Acid (Crystals), 2 grammes. 
Gelatine (Gold label), 6 grammes. 
1 new laid Egg. 

Soak the gelatine in water for at least twelve hours. 
Pour off the water, and place it in a vessel with the 
distilled water. Melt it in a water bath. When 
melted, add the glycerine and acid. Remove the 
vessel from the water bath, beat up the white (only) 
of the egg, and thoroughly incorporate it with the 
fluid at a temperature of from 30 to 35 C. (not more). 
Replace the mixture in the water bath, and gently 
boil it until it has assumed a flaky appearance. 
Now filter it through the finest filtering paper, 
which must be done either in a warm oven, or with 
a hot filter. Keep it in a well stoppered stock 
bottle, and place some, for use, in a wide mouthed 
capped bottle. When used, the bottle should be 
placed in a bath of water sufficiently heated, and 
kept so, to thoroughly liquify the " Jelly." 



1 Carbolic Acid (Crystals), or Formic Acid may be substi- 
tuted for this ingredient the former, indeed, is generally 
used but Boracic Acid does not affect hsematoxylin stainings, 
which are unquestionably sooner or later much injured by the 
other acids. 



ON STAINING FLUIDS, AND STAINING. 101 

2. LAWRENCE'S FORMULA. 

Soak J Ib of best gelatine for some hours in cold 
water, pour off the water, and heat the gelatine 
until it melts. Let it cool, but whilst it still 
remains liquid, add to each (fluid) ounce a (fluid) 
drachm of white of egg. Boil gently until it 
becomes clear, filter it through fine flannel, and 
add to each ounce of the solution 6 drachms of a 
mixture of 1 part (Price's) Glycerine and 2 parts 
of Camphor water. 



3. KAISER'S FORMULA. 

Place one part of finest " gold label " French 
gelatine in 6 parts of distilled water, let it soak for 
two or three hours, then add 7 parts of best glyce- 
rine, and for every 100 grammes of this mixture, 
L gramme of strong Carbolic acid. Warm for from 
ten to fifteen minutes, stirring all the time ; when 
all the flakiness has been removed, filter it. 



GELATINE AND HONEY. (JJeane's medium). 

fy Gelatine (Best " gold label "), 1 ounce. 
Honey, 5 ounces. 
Distilled water, 5 ounces. 
Eectified alcohol, | ounce. 
Creasote, 6 minims. 

Soak the gelatine in the water until it is quite 
softened. Boil the honey in a water bath, and 
when it is boiling add the gelatine and water. 
Now boil the mixture, let it cool slightly, then 
mix the alchohol and creasote together and add 
them. Filter through fine flannel. 



102 METHODS OF MICROSCOPICAL RESEARCH. 

GUM AND GLYCERINE. (Warrant's medium). 1 

1. Gum arable (Best picked), 4 ounces. 
Distilled water, 4 ounces. 
Glycerine, 2 ounces. 

Keep it in a well stoppered bottle with a lump 
of camphor in it. 

2. Gum arable, 1 ounce. 2 
Glycerine, 1 ounce. 
Distilled water, 1 ounce. 
Arsenious acid, 1| grains. 

Dissolve the arsenious acid in the water, and the 
gum in this mixture, without heat, add the glycerine 
and incorporate with great care to avoid the forma- 
tion of air-bubbles. 

Glycerine, mixed with distilled water, forms an 
admirable medium in which to examine tissues and 
specimens and make minute dissections. It is 
sometimes used thus diluted as a mounting fluid, 
but is not to be relied upon as a preservative 
medium, whereas undiluted glycerine, when once 
the crucial difficulty of sealing it up and preventing 
leakage from the preparation has been successfully 
achieved, may be considered not only unsurpassable 
as a mounting fluid in cases for which it is suitable, 
but entirely reliable as a preservative. The her- 
metically sealing of the glycerine under the cover, 
or in the cell, must be most carefully done by 
applying at least three coats of shellac cement to 
be followed by two or three coats of white zinc 
cement, the formulae for making which will be given 
in their places, and the method of applying them 



1 Beale, page 68. 2 Micrographic Dictionary. 



ON STAINING FLUIDS, AND STAINING. 103 

described in the chapter on Mounting. All 

tissues, sections, and specimens, to be mounted in 
pure glycerine, should be subjected to a prolonged 
immersion in glycerine and distilled water, the pro- 
portion of the glycerine to that of the water being 
gradually increased every day or two until pure has 
been substituted for diluted glycerine. Great care 
must be taken that the specimen shall be thoroughly 
saturated with the glycerine before mounting it 
therein. 

This soaking may be done either in watch-glasses 
or very small staining saucers, and many specimens 
or sections can thus be immersed together. In 
order to prevent the glycerine from absorbing 
moisture from the atmosphere, and to ensure clean- 
liness, the saucers should be placed under bell- 
glasses whilst the soaking is in progress. This 
process, moreover, prevents the serious shrinkage 
which too frequently results, especially with delicate 
tissues and specimens, which have been mounted in 
glycerine without previous saturation therewith. 
Tissues, sections, delicate marine animals, insects, 
&c., which are to be mounted in glycerine may be 
preserved therein until required. 

Glycerine undoubtedly affects logwood stainings 
adversely, but if slightly acidulated with acetic acid 
it does not attack 1 carmine or picro-carmine. 

Acetate of Potash. Make a saturated solution 
in distilled water, it may be so used or diluted if 

1 Farrant's medium is preferable for the mounting of speci- 
mens stained by either of these. It is advisable to rather 
overstain and to soak the specimens in the medium for two 
or three days, in order to extract the superfluous stain before 
mounting them. 



104 METHODS OP MICROSCOPICAL RESEARCH. 

necessary. This fluid should be allowed to flow 
under the cover by capillary attraction, and the 
mount be sealed up in from twenty-four to thirty- 
six hours afterwards. 

NAPTHA AND CREASOTE. (Dr. Beale's formula.) 

Creasote, 3 drachms. 
Wood-naptha, 6 ounces. 
Distilled Water, 64 ounces. 
Chalk, quant, suff. 

Mix the naptha and creasote together, and add 
sufficient chalk to produce a thick paste. When this 
has been mixed smooth, add gradually the water, 
thoroughly incorporating all the ingredients in a 
mortar, and let it stand in a lightly covered vessel 
for two or three weeks. Stir it occasionally. Then 
pour off the super-natant fluid, which, if necessary, 
filter. Finally, add some small lumps of camphor, 
and keep it in a well-stoppered bottle. 

GOADBF'S FLUID. 
Bay Salt, 4 ounces. 
Alum (powdered), 2 ounces. 
Corrosive Sublimate, 4 ounces. 
Distilled Water, 2 quarts. 

This is an admirable preservative for delicate 
marine specimens, all entoinostraca and allied 
organisms ; for larvae and some entomological 
specimens. 

Mounting Fluid for Algfie (of which it preserves 
the colour) and for fresh gatherings of diatomacese, 
desmidiaceae, &c. 

Acetate of Copper, 15 grains. 
Camphor Water, 4 ounces. 
Distilled Water, 4 ounces. 
Acetic Acid (glacial), 20 minims. 



ON STAINING FLUIDS, AND STAINING. 105 

Mix and add 

Glycerine (Price's best), 8 ounces. 
Bichloride of Mercury, 1 grain. 

Mix thoroughly, and filter very carefully, 
Sty rax. A liquid gum which exudes from the 
bark of the Liquidambar orientate. This is 
used specially for the mounting of diatomaceae, and 
the method of its use will be described when, in the 
chapter on " Mounting," full instructions are given 
for cleaning and mounting these organisms. Styrax 
is soluble in alcohol, ether, benzol, and xylol, which 
last is its best solvent for microscopical purposes. 
The solution should not be too thick, and must be 
carefully filtered. Styrax is preferable to Canada 
Balsam as a medium in which to mount diatoms, 
its refractive index being greatly higher than that 
of Balsam, whilst it is equally safe and durable. 

Monobromide of Naphthalin is soluble in 
alcohol and ether ; its high refractive index- 
double that of Canada balsam renders it a valu- 
able mounting medium for diatomacese. The cover 
should be " ringed " with wax before any cement is 
applied to secure it. Then two coats of shellac 
varnish, followed by white zinc cement, or, if the 
slides are to be used with oil immersion objectives, 
by the special cement of which the formula is given 
amongst the 

CEMENTS. 

Dammar Varnish. Gum dammar, dissolved 
in benzol, has been much used as a mounting 
medium ; its only recommendation, however, is 
that it is easy to apply. It is not safe, neither is 
it durable, and the author strenuously advises that 



106 METHODS OF MICROSCOPICAL RESEARCH. 

it shall be entirely discarded for all purposes of 
mounting, and used only as a cement, in which 
character it has its uses, and especially it is good 
for " ringing " glycerine jelly and glycerine 
" mounts " before the application of the shellac 
varnish, and the finishing white zinc cement. 
Dammar varnish is made thus : 

1^ Indian Gum Dammar, 4 drachms. 

Benzol (best) 8 (fluid). 

Let it stand for two or three days, then pour off 
the super-natant liquid, and add thereto 1 drachm 
of spirits of turpentine. Filter if necessary. 

Bell's Cement. 1 This is a thoroughly reliable 
cement for many purposes ; it is easy to use, as it 
flows readily from the brush and hardens rapidly. 
Shallow cells can be made with it, and it may be 
used for sealing up preparations mounted in fluids, 
glycerine, or glycerine jelly, in place of shellac 
varnish, the cover being previously " ringed" with 
the following : 

GELATINE CEMENT. (Marsh's formula.) 

Soak half an ounce of gelatine in water until it is 
thoroughly soft. Pour off nearly all the water, and 
melt the gelatine in a hot water bath ; add 3 or 4 
minims of creasote and keep it in a capped bottle. 
It must be used warm, it sets very quickly, and may 
be followed by Bell's cement, or shellac varnish^ 
and the slides be finished with white zinc cement. 

1 This cement must be purchased from its makers, Messrs. 
J. Bell & Co., 338, Oxford Street, London, or from the 
opticians ; its formula being a secret. 



ON STAINING FLUIDS, AND STAINING. 107 

Asphalt Varnish. It is better to purchase 
this also. Mr. Kitton, however, finds that asphalt 
dissolved in benzol, and to which is added some 
gold size, answers its purpose well. 

Qold Size. This, again, should be purchased, 
as also should Marine Glue, which, as well as the 
following cement, is used for affixing cells to slips. 

G-UTTA PEECHA CEMENT. (Harting's formula.) 

Divide gutta percha into small pieces, and dis- 
solve it, by means of moderate heat, in 1 5 parts of 
oil of turpentine ; when dissolved, strain it through 
linen, and whilst the solution is kept at a gentle 
heat, stir into it one part of shellac. 

For affixing cells to the slips, heat the cement, 
make a ring on the slip the size of the cell, attach 
the cell and heat the slip until the cell is affixed by 
an even layer of the cement, so that no leakage may 
occur. 

Shellac Cement or Varnish. Break up 
shellac into very small flakes, and put these into 
a bottle with strong methylated spirit ; shake it up 
frequently, until there results a solution sufficiently 
thick to be used easily with a brush. When from 
keeping it becomes too thick, add more spirit. 

WHITE ZINC CEMENT. 
(Walmsley's formula), and given by him to the author. 

In order to ensure that the white oxide of zinc 
which is the foundation of this valuable and per- 
manent cement shall be of the best quality, and 
well ground, it is advisable to purchase a large tube 
of artist's "zinc white," rather than to grind for 



108 METHODS OF MICROSCOPICAL RESEARCH. 

oneself, the ordinary white oxide of zinc in drying 
oil. The tube of zinc white is to be emptied into a 
large bottle, and as much as possible of the oil to be 
removed, by shaking up the white zinc in a con- 
siderable quantity of benzol, and when the zinc has 
settled to the bottom of the bottle, pouring it off. 
A saturated solution of gum dammar in benzol is 
to be made in another bottle, which should be 
placed in a water bath, kept hot, until the gum 
dammar is dissolved. "When this is accomplished 
the solution of gum dammar is to be poured upon 
the white zinc, the bottle thoroughly shaken up, 
and the mixture strained through fine muslin. Now 
add about half a drachm of best gold size to each 
ounce of the solution. If too thin, allow the benzol 
to evaporate until the cement becomes of a con- 
sistency to flow smoothly and readily from the 
brush if too thick, add benzol. This cement 
should be kept for use in a capped bottle with a 
wide mouth, and thoroughly stirred (not merely 
shaken) up each time it is used. White zinc cement 
properly made according to this formula, is tho- 
roughly reliable and safe. It never cracks, will 
stand ordinary wear and tear well, but not, of 
course, rough usage. The author's experience is, 
that no other white zinc cement than this is of any 
value or permanency. Preparations intended for 
use with oil immersion objectives must not be 
finished with this cement, as the essential oils used 
for immersion purposes at once attack and soon 
destroy it. 

Cement for finishing preparations to be 
examined with immersion objectives. 

Place five or six pieces of gum mastic, about the 



ON STAINING FLUIDS, AND STAINING. 109 

size of a pea, in sufficient strong methylated spirit 
to dissolve them thoroughly. 

Soften some isinglass in water, remove all the 
water and dissolve it in two ounces of absolute 
alcohol. 

To this add two small pieces of gum ammoniacum 
rubbing them up in a mortar until they are dissolved. 

Mix the two solutions, and keep the cement in a 
wide mouthed stoppered bottle. 

When using it place the bottle in hot water until 
the cement is liquefied. 

Embedding and Freezing. 

The great desideratum in embedding specimens, 
for the purpose of making sections of them, is, that 
they shall be firmly and equably supported in all 
directions and that no distortion, displacement or 
injury to the tissues, organs or organisms embedded 
shall result. It is therefore most important that 
the student should acquire the knowledge and 
experience necessary to enable him to decide which 
process will afford and ensure the delicacy combined 
with solidity required in special cases. 

The methods of embedding are twofold, " simple " 
and " interstitial," the former consisting in enclosing 
the specimen to be sectionised in a soft substance 
which will consistently and equably support it in all 
directions ; whilst the latter involves the careful 
infiltration of the specimen with, and its thorough 
permeation by, a " mass " which, whilst warm, is 
liquid, and which solidifies as it cools ; or, as in 
the case of celloidin, the saturation of the specimen 
with that substance, held in solution by ether, 



110 METHODS OF MICROSCOPICAL RESEARCH. 

which evaporating leaves the specimen perfectly 
embedded in a transparent and firmly gelatinous 
matrix. 

Lee, in his invaluable Text-Book 1 treats of the 
"theory of embedding" in a manner so exhaustive 
and practical that the author ventures to quote 
largely from his clever pages and tendering his best 
acknowledgments. Lee, then, says, after referring 
to the simplest methods of embedding : " A further 
object is proposed in the case of the other (i.e., the 
more elaborate) class of methods, which may be 
designated methods of interstitial embedding or 
infiltration methods. In these it is proposed to fill 
out with the imbedding mass the natural cavities of 
the object, in order that their lining membranes, or 
other structures contained in them, may be duly 
cut in situ, or, going a step further, it is proposed 
to surround with the supporting mass not only each 
individual organ, or part of any organ that may be 
present in the interior of the object, but each 
separate cell, or other anatomical element, thus 
giving to the tissues a consistency they could not 
otherwise possess, and ensuring that in the thin slices 
cut from the mass all the details of structure will 
precisely retain their natural relations of position. 
Such a process of embedding is at the same time 
practically a process of hardening, in so far as it 
enables us to give to tissues a degree of firmness 
that could otherwise only be obtained by the em- 
ployment of chemical processes, such as prolonged 
treatment with chromic acid and the like. The 



1 "The Microtomist's Vade Mecum " (page 162). J. & A. 
Churchill. 



ON STAINING FLUIDS, AND STAINING. Ill 

principle of the methods of this second class is 
either, (like that of the first,) that by the immersion 
of the object to be cut in some material that is 
liquid whilst warm, and solid when cold, all the 
parts of the object may be duly surrounded by the 
supporting mass (the second class differing from the 
first chiefly in the employment of materials possess- 
ing greater power of penetration whilst liquid, in 
longer immersion in the liquid mass, and in such 
previous preparation of the object, by soaking in 
some liquid that is a solvent of the embedding 
material, as makes it more readily susceptible of 
infiltration by the latter) ; or the processes may 
be based on another principle, namely, that of the 
employment of substances which whilst in solution 
are suniciently fluid to penetrate the object to 
be embedded, whilst at the same time after the 
evaporation or removal by other means of their 
solvent, they acquire and impart to the embedded 
object sufficient firmness for the purpose of cutting. 
The collodion process suniciently exemplifies this 
principle. If a piece of soft tissue be dehydrated, 
and soaked first in ether and then in collodion, and 
if the ether be allowed slowly to evaporate, the 
tissue and surrounding mass of collodion will 
acquire a consistency such as to admit of thin 
sections being cut from them." 

Embedding in Carrot. This is a very 
primitive and almost exploded process, but it has 
its uses and advantages when cylindrical specimens, 
for instance, vegetable stems, are to be embedded ; 
it is also available for such tissues as can be 
rolled up and so enclosed in the centre of the 
carrot. A " well " microtome is, of course, neces- 



112 METHODS OF MICROSCOPICAL RESEARCH. 

sary to this process, whilst a set of cork cutters, 
or of brass tubes sharpened at one end, must be 
provided ; one of these tubes must be of the 
exact diameter of the well of the microtome, and the 
others, progressively, of smaller diameters, so that 
the various diameters of the specimens may be 
matched. A sound carrot must be chosen and a 
cylinder cut out of its centre, with the largest tube, 
precisely to fit the well of the microtome, and of the 
same length as the depth of the well ; now, with a 
knife divide this cylinder into two equal halves, 
place the two halves together, and with the tube 
nearest in size to, but slightly smaller than, the 
specimen to be embedded cut out the exact centre 
of the cylinder; this will give two semi-circular 
pieces of carrot each with a semicircular groove, 
in one of which place the specimen, lay the other 
piece upon it, and the specimen is embedded in a 
cylinder fitting tightly into the well of the microtome, 
and sections can be cut, after practice, of a great 
degree of thinness, and without difficulty. 

Embedding in Pith. This substance is used 
in the same manner as the carrot. A cylinder of 
pith exactly the size of the well of the microtome 
is divided longitudinally into two equal halves, and 
the specimens embedded between the two halves, 
and if possible without the removal of any of the 
central pith. Pressure and "kneading" will 
generally be found sufficient to form a central tube, 
in which the specimen can be enclosed, with the 
advantage that no portion of the pith having been 
actually removed, the cells displaced by pressure 
will to a great extent recover their original position 
and exert the desired central pressure and give 



ON STAINING FLUIDS, AND STAINING. 113 

the necessary " all round" support to the specimen. 
The embedding being accomplished, the cylinder is 
to be driven into the well of the microtome and 
moistened with alcohol, which will cause the pith 
cells to swell and firmly enclose and support the 
specimen, whilst if the cylinder should not already 
fit into the well with the necessary exactitude this 
swelling will ensure perfect tightness. 

Wax and Oil. Equal parts of white wax and 
olive oil are to be melted by heat over a sand bath. 
In warm weather, or in a hot room, the proportion of 
wax should be slightly increased ; in colder weather, 
or in a cool room, increase the proportion of oil. 
The mass must, of course, be melted when used. 
The specimens to be embedded should be immersed 
in alcohol and thoroughly dehydrated, and then 
placed for a few seconds in collodion, which being 
allowed to evaporate to dryness, the specimen is 
then embedded either in the well of the microtome 
or in a paper tray. The ring of collodion surround- 
ing the sections will be dissolved away by the clear- 
ing medium. 

Embedding in Paraffin. The infiltration 
with paraffin, and subsequent embedding of speci- 
mens therein, is a process of much nicety and 
requiring great care, and first, the selection of a 
suitable paraffin and the composition of the "mass" 
are all-important. Two paraffins with the different 
melting-points of 110 F. and 140 F. should be 
combined in such proportions as shall give a firm 
and homogeneous mass which, when after melting it 
has solidified, will cut perfectly smoothly in a tem- 
perature of from 55 to 70 F. It will be found in 
practice that two parts of the paraffin of the 140 
8 



114 METHODS OF MICROSCOPICAL RESEARCH. 

melting point, and one part of that melting at 110, 
will afford a mass which will answer perfectly well 
when cut in a temperature of 70 D . That with the 
lower melting point being combined in the propor- 
tion of one-fifth of its bulk with four- fifths of that 
of the higher melting point, will cut well in a tem- 
perature of 60 ; whilst equal parts of the two 
paraffins at 110 and 140 melting points respectively 
will give the most satisfactory results when cut in 
a temperature of 55 F. 

The paraffin is to be melted, in a water oven, in 
porcelain pipkins (having covers and handles), and 
the embedding may be done in paper or cardboard 
trays, but it will be found more convenient and 
much safer to use, according to the size of the 
specimen to be embedded, the small porcelain pans 
in which moist water colours are sold (these give 
nice little firm blocks requiring no trimming) ; for 
larger specimens, small porcelain vessels used in 
various chemical processes are readily procurable, 
whilst small glass vessels, or large (flat-bottomed) 
test-tubes will answer admirably for specimens of 
any considerable size. 

The specimens must be thoroughly hardened and 
perfectly dehydrated, and when about to be embedded 
are to be placed in strong alcohol, or, better still, in 
absolute alcohol, for a short time ; from this they 
are removed into creasote and left therein until they 
are well impregnated (say for six hours). Having 
removed as far as possible all creasote from their 
surfaces, the specimens are placed in the melted 
paraffin, in which, kept at a temperature slightly 
above its melting-point, they are to remain from 
one to four hours according to their size, and to 



ON STAINING FLUIDS, AND STAINING. 115 

ensure absolute permeation by, and saturation with, 
the paraffin. The blocks must be allowed to cool 
thoroughly before they are removed from the 
moulds; when removed, they may be pared down 
to any size desired, and in such a manner that the 
face of the embedded specimen, from which it is 
intended to cut sections, shall be at the surface. 
A sufficient body of paraffin must be left surround- 
ing the specimen to ensure the necessary solidity 
and equable support. Specimens thus embedded 
and prepared for sectionising may be kept for an 
indefinite time without alcohol, and used when 
required. As they are perfectly embalmed and 
preserved, the only necessary precaution is to 
ensure absolute freedom from moisture in the 
bottles or boxes in which the blocks are stored. 

The paraffin can be dissolved out of the sections 
by means of chloroform, turpentine, turpentine and 
oil of cloves mixed in equal proportions (this will 
also clear the sections), or xylol; the latter being 
of course preferable when the sections are to be 
" cleared" in xylol and absolute phenol, and mounted 
in xylol-balsam. When turpentine (only) is used, 
and the sections are to be cleared in xylol and 
phenol, they must be placed in absolute alcohol for 
a short time to remove the turpentine, before being 
immersed in the clearing fluid. 

Specimens can also be satisfactorily embedded 
in paraffin by the following method (Giesbrecht* s) , 
founded upon the rapid evaporation of chloroform, 
and which, at the same time, is a speedy solvent of 
paraffin. The specimens to be embedded having 
been immersed to saturation in absolute alcohol are 
next placed in chloroform ; a short time having 



116 METHODS OF MICROSCOPICAL RESEARCH. 

been allowed for saturation therewith, the chloro- 
form and the specimens are together heated to the 
melting point of the paraffin, small pieces of paraffin 
being gradually added during the heating ; air 
bubbles will arise from the specimens, and when 
these cease no further addition of paraffin will be 
necessary. The " mass " must be kept at its 
melting-point in a dry oven for some considerable 
time after the addition of the paraffin, in order to 
ensure the entire evaporation of the chloroform, 
any remains of which in the mass would prevent 
its becoming sufficiently hard to cut satisfactorily. 
The embedding is of course done as in the ordinary 
paraffin process. 

EMBEDDING IN GUM AND GLYCERINE. 

Iy Gum Acacise Mucilage, B.P., 90 parts. 
Price's Glycerine 10 ,, 

Add 1 per cent, of carbolic acid crystals. 

Place the specimens to be embedded in this mix- 
ture in watch glasses, or small (water colour) saucers, 
and let them remain exposed to the air of the labo- 
ratory for four days. In that time the mixture will 
have set into a firm, gelatinous mass. Blocks, in 
each of which one specimen is enclosed, are then to 
be cut out, and again left exposed for three days to 
hardeo, being turned over from time to time that 
they may dry equally on all sides. When dry, the 
blocks may be pared down to suitable size, and 
affixed to corks with strong gum water, and on 
these they may be preserved, to be sectionised as 
required. 

The gum can be removed from the sections by 
immersion in distilled water before staining them. 



ON STAINING FLUIDS, AND STAINING. 117 

Embedding in Celloidin. 1 This delicate and 
cleanly and most effective process is pursued as 
follows : 

A thin solution of the celloidin is made in equal 
parts of absolute alcohol and ether. 

A thick solution is also made in the same manner, 
by increasing the proportion of the celloidin. 

The specimens perfectly dehydrated are to be 
placed in absolute alcohol for twenty-four hours, and 
then transferred into a mixture of equal parts of 
absolute alcohol and ether for a second period of 
twenty-four hours. 

They are now to be immersed in the thin solu- 
tion of celloidin for twenty-four hours, and after- 
wards in the thicker solution for three days. 

They are then to be placed in paper trays, so 
made as to admit of a good margin of celloidin sur- 
rounding the specimen occupying the centre of the 
tray. The tray is then to be filled with the thick 
solution of celloidin, which is to be allowed to 
evaporate, the tray being filled up from time to 
time, as evaporation goes on, until the specimen 
is covered, as well as surrounded, by a layer of 
celloidin in other words, embedded in a block of 
celloidin, of the size and shape of the tray. When 
the celloidin begins to set firmly, and before any 
shrinkage sets in, the tray, with the specimen, is 
to be plunged into a vessel with an air-tight cover, 
containing strong methylated spirit, and in this the 



1 " Schering's Celloidin," which is patented, can be obtained 
at Mr. C. Baker's, 244, High Holborn, London, W.C. The 
celloidin shavings, recently introduced, will be found much 
more convenient to use than the tablets. 



118 METHODS OP MICROSCOPICAL RESEARCH. 

blocks may be preserved until required for section- 
ising. The purpose, in embedding in celloidin, is 
to secure the normal position (in situ) of all the 
component parts of an organism or specimen. The 
film of celloidin surrounding all the organs and 
parts, and preserving their respective features and 
positions, must be retained, and mounted with the 
section. When these sections are to be mounted in 
Canada balsam, they must be cleared in xylol and 
phenol (as oil of cloves destroys the celloidin) and 
mounted in xylol balsam. Celloidin sections may 
be mounted in glycerine, glycerine jelly, or Farrant's 
medium, all of which render the celloidin nearly as 
transparent as does balsam. These sections may 
be stained by means of all the ordinary staining 
fluids, except, perhaps, one or two of the more 
powerful anilin dyes which sometimes, though 
curiously enough not invariably, colour the cel- 
loidin indelibly. If stained by logwood they should 
be strongly stained in order to admit of the extrac- 
tion of the colour from the celloidin by the acetic 
acid process, whilst the tissues remain sufficiently 
stained. 

Specimens embedded in celloidin should be cut 
by the ether freezing process. 

Gum and Syrup. For ordinary tissues 
make a mixture of 5 parts of gum mucilage B.P. 
and 3 parts of simple syrup B.P. For nerve, 
brain, spinal cord, and any brittle tissues the com- 
bination should be 5 parts of gum mucilage to 4 
parts of syrup. 

Tissues and specimens to be cut upon the ether 
freezing microtome are to be immersed in this fluid 
for three or four days. They may be preserved for 



ON STAINING FLUIDS, AND STAINING. 119 

an indefinite time in the mixture, indeed, perma- 
nently. The specimens must have been carefully 
hardened and thoroughly freed, by prolonged soak- 
ing in water, from all traces of the hardening 
reagents, before placing them in the gum and syrup. 
The ether freezing process is conducted thus. 
The gum and syrup is wiped off the specimens with 
a cloth, and they are placed in the strongest possible 
solution of gum acacias for a few hours before being 
frozen and cut. The microtome and freezing ap- 
paratus having been prepared for use, a specimen 
is placed upon the freezing plate and painted round 
the edges and over the surface by means of a brush 
with the strong, pure gum-water from which it was 
taken. It is then frozen by means of the ether 
spray until it can be easily and smoothly cut. 
Practice and experience will soon enable the 
operator to stop the freezing at the right moment. 
Should the ice, however, become too hard, and 
therefore brittle, a few seconds' delay before com- 
mencing to cut the sections will ensure its being 
in the right condition for cutting. 

On Section Cutting and Microtomes. 

Section cutting, like staining, is an art requiring 
not only practice and experience, but knowledge 
and judgment. Thorough knowledge of the struc- 
ture and functions of the organisms, organs and 
specimens to be sectionised is equally necessary with 
the judgment enabling one to decide as to the 
thickness or thinness of the sections of, say, an 
organ which shall most satisfactorily display its 
structure. It is quite possible to cut sections too 



120 METHODS OP MICROSCOPICAL RESEARCH. 

thin so thin, indeed, as to obliterate all the features 
and structure which it is necessary to study in order 
to arrive at the physiological or biological elements 
of the specimen. Brain, spinal cord, and the like 
tissues, for example, cannot be cut too thin kidney 
and several other organs can easily be so, and thus 
be rendered worthless for purposes of study. It is 
well, therefore, to provide one's self with three 
sections of such specimens, as it may be found 
possible, by experience, to cut too thin ; so that for 
general observation and study, with low powers, a 
thin section is at hand, for medium powers a thinner 
section, and the thinnest for use with high and the 
highest powers. 

Some writers and practical workers assert that 
it is not possible to cut the thinnest sections from a 
celloidin-embedded specimen by the ether freezing 
process ; the author can only say that he has cut 
sections of entire human eyes, so embedded and 
frozen, sireoths of an inch thick (or rather thin), 
and of brain and spinal cord, &c., jcfeoth of an inch 
thin. 

For cutting sections by the ether freezing process 
there is (with the exception of the automatic Minot 
microtome recently produced and fully described in 
later pages) no better microtome than the Cathcart, 
as perfected and supplied by Mr. Alexander Fraser, 
of Edinburgh (which is fully described and illustrated 
on pages 127-8), with admirable arrangements and 
apparatus for embedding specimens in paraffin and 
for cutting those also, the freezing and this arrange- 
ment being removable at will and interchangeable. 



ON STAINING FLUIDS, AND STAINING. 121 

Professor Rutherford's Microtome. 

This is Stirling's original well and screw- 
microtome, plus an ice box arrangement. It is 
used by filling the ice box with well-powdered and 
mixed ice and bay salt in equal quantities. Whilst 
freezing is going forward the well may be covered 
with felt. One or several tissues can be cut at 
once, either with a Rutherford's knife, or, what is 
now found to be far better, a plane iron. By a 
plane iron, we mean the iron taken out of a car- 
penter's smoothing plane. These irons must be 2f 




inches broad, and are used either with or without a 
covering of wood. When double-faced with wood, 
the handling of them is more pleasant, but it does 
not add to efficiency. The iron must be sharpened 
on a very smooth hone, and then well stropped. 
In sharpening, care must be taken to rub the entire 



122 



METHODS OF MICROSCOPICAL RESEARCH. 



length of the cutting edge evenly. This is best done 
by taking care to rub the edge on the stone in such 
a way that every portion of its length is upon the 
stone at the same time. In stropping, it will be 
found most convenient to rest the plane iron against 
a table edge, and push or pull the strop over it. 

The advantage of the Rutherford freezing micro- 
tome is that it can be also used as an ordinary 
Stirling's microtome for imbedding in carrot, paraffin 
or wax. Its disadvantage is the same as that of 




ice freezing machines of whatever description that 
it is cold, disagreeable work filling in the ice and 
salt, and the former is not always to be obtained, 
and when once charged, cutting must follow, or the 
ice melts. 

Mr. C. Baker, 244, High Holborn, W.C., sells an 
excellent Rutherford's freezing microtome, which, 
of course, must have its cutting face covered with 
plate glass. 



ON STAINING FLUIDS, AND STAINING. 123 

Williams' Freezing Microtome. 

This is made by Messrs. Swift & Son, 81, Totten- 
ham Court Road, London, W.C., who also make a 
registered knife carrier, which carries^the blade of a 
razor : the whole goes under the name of " Swift's 
Knife." Until the plane iron was thought of, 
Swift's Knife had the field all to itself, and there 
will still be those who will prefer it to the plane 
iron. 




This combination has this great advantage : that 
in winter, when ice is plentiful, the machine is 
charged, and freezes strongly for an liour or more. 
For class work this is excellent : the attendant 
charges the machine just before the class meets, 
and the demonstrator cuts a hundred sections of 
any prepared tissue in a few minutes. If he wishes 
to cut another specimen, he removes the first, and 



124 METHODS OF MICROSCOPICAL RESEARCH. 

clears out the grooves on the top of the section 
carrier with the back of a knife ; then he places his 
specimen upon the machine, and paints it round 
with gum solution, and the whole freezes fit for 
cutting in one minute or so. With very little prac- 
tice it is quite easy to cut fifty excellent sections of 
each of fifteen or twenty specimens with one charge 
of ice and salt. 

To manage Swift's knife, we proceed thus : 
First take the blade away, and lay it flat upon a 
smooth hone well oiled, and rub it first on one side, 
then the other, until the entire edge on either side is 
ground by the hone at once. The writer rubbed 
over twenty hours before this was accomplished 
with the razor blade of his Swift's knife. Both 
sides of the blade are ground quite flat, to begin 
with, not hollowed ; but cutlers either cannot or 
will not grind a blade with its back and its edge 
perfectly parallel, so that this has to be done by 
one's self. Having ground the knife blade, we fix 
it in the handle and strop it. We then take off the 
handle and fix the blade in the carrier in such a 
way that the edge must be perfectly level with the 
top of the microtome, and on a much lower level 
than the back. This position of the knife in the 
frame is to the last degree essential, and we effect 
it thus : First place the knife-blade in the frame, 
and lower the screws which the edge of the blade 
rests upon, so that the edge is lower than the back 
a good deal. After making it firm, we complete the 
levelling by freezing a piece of tissue, and taking 
away a thick section, cutting away from us, with 
the knife in the frame just as we have put it roughly. 
We now place each end of the blade alternately 



ON STAINING FLUIDS, AND STAINING. 125 

against the same part or bit of the frozen tissue, and 
raise or lower either end by screwing either of the 
two screws nearest to us. All is now complete, and 
we cut section after section by lowering the knife 
edge for each fresh cut by giving the screw furthest 
away from us a part of a revolution. 

To Fill an Ice Microtome. 

Get a piece, or pieces, of ice the size of two closed 
fists, and the same bulk of either common table salt 
or, even better, of bay salt. Powder both, and 
thoroughly mix them. The ice is most readily 
reduced to fine powder by being surrounded with 
flannel and bruised with a broad-faced hammer, 
or a carpenter's mallet. Fill in the mixed ice 
and salt and press it well down, but take care that 
the trough of the microtome is not so full that the 
top touches the ice and salt. The top is to be placed 
on the microtome and screwed fast. In winter the 
machine, after being charged, is ready for freezing 
in five minutes or so ; in summer it may be ten 
minutes or more. Freezing proceeds, as we have 
before said, for an hour or two. The waste pipe at 
the bottom of the microtome is to be connected 
by caoutchouc tubing, with a basin to receive the 
melted ice. 

The disadvantage of Williams' microtome is, that, 
like all ice microtomes, it is disagreeable to charge 
even when ice is plentiful, and cutting must take 
place at the time the machine is charged ; that is to 
say, if the operator is called away, as in medical 
practice often happens, his ice charge melts and 
has to be renewed. 



126 



METHODS OF MICROSCOPICAL RESEARCH. 



Dr. Fearnley's Ether Freezing Microtome. 

Dr. Groves suggested the conversion of Williams' 
ice freezing microtome into an ether freezer. This 
is now called the Grove- Williams microtome, and is 




made by Messrs. Swift & Son, and used with Swift's 
knife. 

For those who are not affected by the inhalation 
of atmospheric air charged with ether, the best and 
simplest ether freezer for use with Swift's knife is 



ON STAINING FLUIDS, AND STAINING. 127 

Dr. Fearnley's ether freezing microtome. This is 
simply the top of the Grove- Williams' microtome, 
fitted with a clamp and screw arrangement for 
attaching it to the work-bench. The ether nozzle is 
immediately under the frame of the glass plate, and 
the bottle of ether stands under the machine on the 
table as shown in wood-cut. Fearnley's microtome 
is also made by Messrs. Swift & Son. 

Cathcart's Microtome. 

This is an ether freezing microtome for use either 
with a stout razor, or with a plane iron. Cathcart's 
invention, used with a plane iron, for cheapness, sim- 
plicity, and efficacy cannot be surpassed, if equalled, 
by any ether or ice microtome we know of. With 
the expenditure of two drachms of ether, the operator 
can cut sixty or seventy sections in almost as many 
seconds, every section being of exquisite thinoess. 
The ether used is methylated ether of 720 s.g. 

The freezing process has already been described. 
This being accomplished, the tissue is elevated to the 
knife by a very small fraction of a revolution of 
the screw with the left hand, whilst the right drives 
the plane iron. The iron must be held with the 
edge far below the level of the rest of the iron, and 
the screw movement and the push of the iron move- 
ment must take place alternately. When a mass 
of sections has accumulated on the iron, it must be 
floated off into a saucer of water. (See illustration 
on page 128.) 

An admirable and most useful microtome is made 
by Zeiss. It can be seen at the depot of, and 
purchased from, his London agent, Mr. C. Baker. 



128 



METHODS OF MICROSCOPICAL RESEARCH. 



The " Kecking Microtome " manufactured and 
supplied by the Cambridge Scientific Instrument 
Company, is in all respects an admirable and most 




THE CATHCART MICROTOME. 



effective machine, and especially for the cutting of 
serial (or ribbon) sections. A full illustrated 
description, as well as directions for its use, can be 
obtained on application to its makers. 



ON STAINING FLUIDS, AND STAINING. 129 

The " Thoma Microtome " adapted equally for 
the paraffin embedding and ether freezing processes, 
is an exquisitely beautiful and reliable instrument. 
The preferable size for ordinary use is that named 
the Thoma B. 2. A verbal description would be of 
but little use, and the author advises the student 
to see one, and this he can (generally) do on applica- 
tion to Mr. C. Baker. Its manufacturer, Mr. R. 
Jung Mekaniker, Heidelberg, will send a full 
(illustrated) description on application. 

Minot's Microtome. 

This beautiful instrument is made in three forms 
(or to use the inventor's expression, " models "). 

In its construction the chief endeavour has been 
to render its various parts as " massive " as possible, 
and to make the adjustments for the various thick- 
nesses of the sections perfectly automatic, whilst, 
at the same time, portability has been considered. 
The thickness of the section is regulated by a special 
toothed wheel arrangement. On the ascent of a 
vertical slide a lever strikes one of the six spokes 
of a wheel, and the ratchet turns the toothed wheel 
of the micrometer screw so that the horizontal slide, 
to which the preparation is attached, is brought into 
contact with the edge of the knife. The six spokes 
of the controlling wheel are of different lengths, 
and are marked 1 to 6, which numbers correspond 
with the number of teeth on the wheel moved at 
each revolution of the driver. 

In Model I. the total number of teeth is 150, and 
as one complete turn of the micrometer screw equals 
0*5 m.m., by means of the spoked wheel sections 
9 



130 



METHODS OF MICROSCOPICAL RESEARCH. 



can be obtained of the respective thicknesses of 
s-io, rio, rib, Ts 9 <nj, and /zjth mm., or of from about 
3 to 20 mikrons. 

In Model II. thinner sections are obtained by 
means of a differential gear. A second wheel is 
used, and the various spokes, 1 to 6, give sections 
of from 1 to 6 mikrons. 

In Model III. the fine adjustment is again modi- 
fied by the use of two ratchets engaging in the 
toothed wheel, one of which is a half tooth shorter 




than the other. The co-operation of this shorter 
ratchet allows further tenuities of section of from 
\ to 1^ mikrons. 

* The advantages and improvements secured by the 
Minot microtome are, briefly, as follow : 

1. The practically indestructible solidity of all its 
parts. 



ON STAINING FLUIDS, AND STAINING. 131 

2. The smooth and even motions of its slides. 

3. Its moderate size and weight and correspond- 
ing portability. 

4. The great range of section thicknesses, from 
to 40 mm. 

5. Its applicability to large sections up to 5 x 5 
cm. 

6. The forward movement of the specimen en- 
tirely above the knife. 

7. The easy adjustment of the specimen in every 
direction. 

8. The production of absolutely fiat and even 
sections, &c., &c. 

Model III. 1 can be used for cutting specimens 
embedded by the celloidin process, for the ether 
freezing process and for " immersed " cutting. 

Vegetable Tissues 

and specimens can be preserved for an indefinite 
time and for sectionising in 1 part glycerine, 1 part 
alcohol, and 1 part water. This, however, applies 
to such specimens as are not readily obtainable, and 
which therefore must be kept in stock. When 
practicable these tissues should be gathered and cut, 
by the freezing process, whilst quite fresh, as in 
this way the best results will be secured and all 
distortion or shrinkage avoided. 



1 This new microtome also can be obtained from Mr. C. 
Baker, 244, High Holborn, London, W.C. 



132 



CHAPTER VI. 



On Mounting. 

UNTIL within the last few years it was considered 
de rigueur to mount all preparations upon the 
ordinary sized slips (3 in. by 1 in.) and the author 
having ventured to produce slides of large sections 
mounted upon correspondingly wide slips, was told 
by one of our most accomplished microscopists and 
workers that if he (the author) persisted in, when 
necessary, increasing the size of his slips, " everyone 
would curse him." That curse, like most others, 
has fallen very harmless, and now we have in 
England, as has long been the case on the Continent, 
preparations of large size mounted upon slips and 
covered with the thinnest glass covers of dimensions 
corresponding with the size of the section or other 
specimen to be mounted. As a rule, however, the 
ordinary sized slip is adhered to, and save in excep- 
tional cases, and under special necessities or cir- 
cumstances, this is for several reasons, and especially 
because the arrangements of the mechanical stage 
of the microscope are made for slides of those 
dimensions, advisable and convenient. 

Thin cover glasses are to be procured cut into 



ON MOUNTING. 133 

circles, squares, oblongs and ovals and of any 
required size ; circular or oval covers will be found 
the most convenient to use, and the neatest in 
appearance; circular covers can be "ringed" by 
the aid of the turn table with any of the various 
protective cements. Cover glasses are supplied of 
three thicknesses, known respectively as No. 1, 
No. 2, and No. 3. No. 3 covers are to be avoided 
as too thick. No. 2 will be found quite thin enough 
for all ordinary purposes. No. 1 must be used for 
all preparations intended for the higher powers. 

An ounce of No. 1 covers should have a fair pro- 
portion of *003 inch glasses in it, still more of "004 
than *005, advancing in proportion from "003 to *006. 
Should an ounce of No. 1 covers contain more than 
a dozen, or at most a score, over *006, it ought to 
be rejected. As the objectives now used are oil 
immersion, or ought to be, for powers higher than 
an eighth the thickness of the cover may be of less 
importance. For instance, Messrs. Powell and 
Lealand's -fs oil immersion lens works through a 
cover of '006 inch perfectly. Almost anything in 
histological work can be seen with an objective of 
a e inch power : only exceptionally do we require 
so high a power as the A inch. We ought, how- 
ever, to use no cover thicker than a '006 inch, if 
possible. There are numerous cover-glass measures. 
Messrs. Ross make an excellent one. 

Mounting in Canada Balsam. 

Many years ago the author discovered the 
following method of using Canada balsam, and 
having made no secret of it, it is generally known 



134 METHODS OF MICROSCOPICAL RESEARCH. 

as Cole's exposure process. It consists in placing 
the specimen to be mounted upon a cover glass, 
applying exactly the quantity of balsam necessary 
to ensure the permanent preservation of the speci- 
men, and that the balsam shall flow to, but not 
beyond the extreme edge of the cover, and then 
allowing the balsam to evaporate for twenty-four 
or thirty-six hours until all the xylol, or other 
solvent, has left it, and the balsam has set hard 
before mounting it on the slip. The author devised 
a set of "pigeon holes," or. rather grooves, made to 
take 3 in. by 1 in. slips, in which the covers could be 
placed, lying upon slips, during the time allowed 
for evaporation. But when the small cabinets, with 
trays, were introduced, he found these to be, prac- 
tically, all that is needed for this purpose. This 
process of mounting is thus effected. A supply of 
thin glass covers of the sizes necessary for the 
specimens to be mounted having been cleaned, and 
the specimens, if necessary, cleared, they are placed 
upon the covers if sections, being floated on to the 
covers from the clearing fluid and carefully spread 
flat upon the covers. The clearing fluid is then 
drained off by holding the cover with its edge 
against a sheet of blotting-paper and the covers, 
if f in. diameter, are placed in pairs upon slips. If 
f in. covers are used, three are placed on each slip, 
of course with the section, or other specimen, 
upwards. The balsam is then applied, and the slips 
with the covers, balsam upwards, placed in a cabinet 
with trays so that no dust can approach them, and 
they are left for twenty-four hours to evaporate. 
Then a very small drop of xylol balsam is placed in 
the centre of each of the mounts in succession ; a 



ON MOUNTING. 135 

slip is heated and the covers are successively turned 
over on to the slips and the balsam is carefully 
"run" by gentle heat until a neat, bevelled edge 
surrounds the cover. The balsam will be found 
quite hard, when cold, and, if necessary, the cover 
can be washed with spirit and any excess of balsam 
removed from its edge and its surface cleaned. 
Never mount specimens or sections in balsam on 
the slip, placing the cover on the liquid balsam and 
trusting to time for its drying. Practically the 
central portion of the balsam, under the cover, 
never dries, and no such slides are really safe or 
permanent. 

When specimens are mounted in cells filled with 
balsam, Xylol-balsam should be used and allowed to 
evaporate before the cover is applied to the cell, 
the cell being filled up from time to time as 
evaporation goes on. In this way specimens can 
be mounted in cells in hard balsam and rendered 
absolutely permanent. 

Mounting in Glycerine. 

Place the slip on a turn table, and run upon it a 
ring of asphalt. The ring should be the size of the 
cover, which, of course, should be a circle. When 
the asphalt is half dry, but still " tacky," place the 
section on the slip, and put upon it a drop of gly- 
cerine ; then lower the cover over it in the usual 
way, and gently press the edge of the cover all 
round, so as to make it adhere to the asphalt. 
Should it not do so at any point, gently warm the 
asphalt by holding the slip over the flame of a spirit 
lamp ; ^then press the cover home. The slide can 



136 METHODS OP MICROSCOPICAL RESEARCH. 

now be dipped in water, and any glycerine that has 
overflowed or been pressed out, washed off with a 
camel-hair brush. After drying it can be ringed 
with shellac varnish followed by white zinc cement. 

Mounting in Glycerine Jelly. 

Specimens to be mounted in glycerine jelly should 
be immersed in distilled water for some time before, 
in order to ensure the removal of all alcohol from 
the tissues, &c. This being done, place a wide- 
mouthed bottle containing glycerine jelly in a water 
bath kept warm, but not too hot, by a spirit lamp 
or a Bunsen's gas burner placed beneath it. Now 
place the section or other specimen to be mounted 
precisely in the centre of the slip, and apply the 
jelly with a small pipette. Then holding a clean 
cover of the necessary size in forceps, steadily and 
gently lower it upon the jelly. The cover should 
touch the slip with its edge furthest from the 
operator and be gradually lowered so as to drive 
out air bubbles, if any, and not to disturb the 
position of the specimen. This is a delicate opera- 
tion, requiring great care and some practice and 
experience. The secret of success is do not be 
sparing of your jelly, apply plenty and you will not 
be troubled with air bubbles. When cold the excess 
of jelly can be easily removed with a soft brush, 
and the slide should be allowed to dry (not rubbed 
dry) and then ringed with shellac cement and 
finished with two coats of white zinc cement. 

Mounting in Farrant's Medium. 

The author has constantly been asked how it is 
that air bubbles are entirely absent from all pre- 



ON MOUNTING. 137 

parations mounted by him in Farrant's medium, and 
has listened sympathetically to the sorrows and 
disappointments of those who find " the difficulties 
of mounting in Farrant's medium insuperable ; " he 
has also read of a clumsy and absurd arrangement 
for passing this medium through a funnel stuffed 
with cotton wool, which is supposed to eliminate 
the air whilst ensuring the agreeable addition of 
cotton fibres to the slide in place of air bubbles. 
He can only say that he has had great practice with 
and experience in the use of Farrant's medium 
during many years, and has found no difficulty 
whatever in getting rid of air bubbles. Here again 
he advises put an abundant supply, with a glass 
rod, on to the slip, place the specimen, which should 
be immersed in Farrant's medium for twenty-four 
hours before mounting it, in the centre of the drop. 
If there are any large air bubbles, as there possibly 
may be, explode them with a red hot needle or push 
them to the margin of the drop, where they will 
disappear at once. If there are any large air 
bubbles in the specimen, remove these also with a 
needle ; small bubbles will disappear by absorption 
within a day or two. Finally, do not press down 
the cover too much afc first so as to cause too great 
an exudation, from under it, of the medium ; leave 
it resting lightly upon the specimen for a few days 
during which all bubbles, if any, will go, and the 
medium will " set " to some extent ; then press 
down the cover, and the medium having so " set " 
will not again be drawn under the cover by capillary 
attraction to anything like the extent that it is 
when first applied ; and this pressing down of the 
cover can be repeated several times at intervals of 



138 METHODS OF MICROSCOPICAL RESEARCH. 

days, and at length the excess of the medium can be 
washed off with a soft brush, the slide, as in the 
case of the glycerine jelly, be allowed to dry, ringed 
with shellac varnish, and finished with white zinc or 
asphalt. 

Specimens to be mounted in aqueous media, such 
as carbolised water, camphor water, glycerine and 
water, &c., should be immersed for some hours in 
distilled water, and mounted, from that, in a shallow 
cell as already described in the case of glycerine. 



The Preparation and Mounting of Insects. 

There are several processes for preparing and 
mounting insects, each of which possesses special 
advantages, in respect of the results and appear- 
ances it may be desired to obtain. Whenever it is 
possible, and except for special purposes and in 
dissections, insects should be mounted " without 
pressure," since the flattening them out under 
pressure results in distortions, displacement of the 
parts and organs, and unnatural, and therefore false 
appearances. It is, however, impossible to mount 
all insects, or their parts, without pressure, and the 
following process will give good results : 

Place the insect in pure liq. potass, mixed with i 
ammonia fort. The insect must not be allowed to 
remain too long in this solution, and must be tested 
from time to time by placing it in water and press- 
ing the thorax. When the thorax is soft and the 
legs flaccid, immerse the insect in water for from 
fifteen to twenty-four hours, then soak it in glacial 
acetic acid and glycerine (half and half) for some 



ON MOUNTING. 139 

hours. 1 Again , immerse it in water for from twelve 
to twenty -four hours ; it is now ready to be laid out 
upon the slip. Having done this, preserving the 
natural position of the parts as closely as possible, 
place a cover over the insect and tie lightly round 
it with soft cotton, stand the slip on end to drain, 
plunge the slip into a turpentine bath and leave it 
until all moisture has been driven out and the insect 
is thoroughly permeated by the turpentine, drain off: 
the turpentine, apply blotting paper to remove any 
excess of turpentine from under the cover, and run 
in " xylol balsam" by capillary attraction. The 
cover should never be lifted or allowed to shift its 
position after the insect has been laid out. 

When it is desired to preserve the brilliant or 
delicate colours of an insect, it should be placed, 
immediately after being killed by means of chloro- 
form, in liq. potass, without any mixture of ammonia, 
for from two to three days, then soaked in water 
for twenty-four hours, then placed in water with 
ten drops of muriatic acid to each ounce of water, 
for twenty-four hours; it should then be laid out 
and immersed in turpentine and treated as already 
described. 

In order to mount insects, or their parts, " with- 
out pressure," it is generally necessary to adopt 
only the following simple process : Soak the insect 
for two days in equal parts of ordinary alcohol and 
water, after which transfer it to absolute alcohol for 

1 Insects removed from liq. potass., after being soaked in 
water for twenty-four hours, should be kept in strong or 
glacial acetic acid and glycerine (half and half) until required 
for mounting. 



140 METHODS OP MICROSCOPICAL RESEARCH. 

two days, immerse it in turpentine until it is not 
only completely permeated therewith and all air 
removed, but let it remain until it is sufficiently 
bleached or decolourised in other words, rendered 
transparent and in order to ensure this result it 
should be placed in a strong light. Select a cell of 
the requisite depth, of pure tin or vulcanite, which 
affix to the centre of a slip with the French liquid 
glue ; when this is dry, having cleaned the interior 
of the cell, place within it the insect, and fill the 
cell with fairly thick balsam until it presents a 
slightly convex surface above the cell, lay it aside, 
as previously recommended, under cover for twelve 
to twenty-four hours, so that all air may escape, put 
a minute drop of fresh balsam upon the surface of 
the balsam which fills the cell, and carefully place a 
cover, slightly warmed, upon it, close the cell by 
gentle and equable pressure, at once remove, by 
means of a soft brush and benzol, all balsam which 
has exuded under the pressure, let the "mount" 
harden for a day or two, and then apply white zinc 
cement or asphalt. 

Cells are to be obtained of various sizes, having 
pure tin caps to fit exactly over them after the 
cover is applied, and these not only impart great 
strength and security to the cell, but ensure the 
neatest possible finish to the mount. Mr. F. Enock, 
whose exquisite entomological preparations are 
altogether unrivalled, is to be credited with this 
admirable device, as well as with various other 
improvements in the special branch of the art to 
which he has devoted himself with such singular 
success. Insects may be mounted "without pres- 
sure " in suitable cells filled with pure glycerine 



ON MOUNTING. 141 

instead of balsam, and for such preparations Mr. 
E nock's capped cells will be found invaluable. 
Insects mounted in glycerine must, of course, be 
previously prepared by dehydration, in spirit, after 
the potass, process, and subsequent prolonged 
immersion in glycerine until complete saturation 
results. 

The Preparation of Vegetable Sections. 

Whensoever possible, stems, leaves, roots, petioles 
and wood should be cut fresh and sectionised as 
soon as may be ; all such specimens should be kept 
in a mixture of equal volumes of alcohol, glycerine, 
and water. Sections also may be stored in this 
mixture. Nearly all vegetable sections require 
bleaching before being stained ; very delicate 
tissues may be bleached by means of alcohol ; hard 
and deeply-coloured stems and woods must be 
bleached in a liquid thus prepared : 

To one pint of water add two ounces of fresh 
chloride of lime ; shake this up thoroughly two or 
three times, and allow it to stand until the lime 
shall have settled. Make a saturated solution of 
common " washing soda." Pour off the supernatant 
fluid from the chloride of lime, and, by degrees, add 
to it the soda solution, until all precipitation ceases. 
Filter the solution, and keep it in a stoppered bottle 
in the dark. No fixed time can be given for the 
bleaching process, the colour and density of tissues 
being so variable. Experience, however, will be 
rapidly gained, and over-bleaching easily avoided. 
The sections being bleached, must now be washed in 
distilled water, several times changed, and allowed 



142 METHODS OF MICROSCOPICAL RESEARCH. 

to remain for twenty-four hours in the final water, 
to which add 8 or 10 drops of nitric acid to each 
half pint. Transfer the sections to alcohol for an 
hour before staining them. To bring out details of 
structure and to display cell walls, &c., there is no 
better stain than logwood, and for singly stained 
vegetable sections it is to be recommended in pre- 
ference to all other staining fluids. The following 
process will give admirable results : 

1. Remove the section from alcohol to water for 
a few minutes. 

2. To 3 per cent, alum solution for ten minutes. 

3. Stain in aqueous logwood stain. 

4. Place in alum water to remove stain from 
surfaces. 

5. "Wash thoroughly in water. 

6. Dehydrate in strong alcohol. 

7. Clear in xylol and phenol. 

8. Mount in xylol balsam. 

The Double Staining and Mounting of 
Botanical Sections. 

1. Bleach the sections for six hours in the liquid 
already described. 

2. Soak them in water, to be several times 
changed, for twenty-four hours. 

3. Place them, for twelve hours, in the following : 

MORDANT SOLUTION. 

(a) Sulphate of Aluminia, 10 grains. 

Dissolved in Distilled Water 200 grains. 
(6) Acetate of Lead, 30 grains. 

Dissolved in Distilled Water, 600 grains. 

Add a to b until precipitation ceases, then allow 
the whole to settle and syphon off the clear liquor, 



ON MOUNTING. 143 

which must be filtered and kept in a stoppered 
bottle. When required for use dilute a portion 
with four or five additional equivalents of water 
and filter afresh. 

4. Stain in borax carmine solution for one to two 
hours. 

5. "Wash in acidulated water (1 drachm nitric acid 
to 1 pint of water). 

6. Wash quickly in pure water. 

7. Place in alcohol for one hour. 

8. Place in green stain for one to three hours. 

Acid Green, 2 grains. 
Distilled Water, 3 ounces. 
Glycerine, 1 ounce. 

Mix the glycerine and water well together and 
dissolve the green crystals in the mixture, filter and 
keep in a stoppered bottle. 

9. Wash quickly in absolute alcohol. 

10. Clear in xylol and phenol. 

11. Mount in xylol balsam. 

Another Process for Vegetable Sections. 

1. Place the section in an alcoholic solution of 
iodine green (3 grains to the 1 ounce of alcohol) for 
one or two hours. 

2. Soak it in alcohol for ten minutes. 

3. Remove it to water for one minute. 

4. Immerse it for two hours in carmine fluid, 
made as follows : 

Carmine, 15 grains. 
Ammonia, 15 grains. 
Water, 2 ounces. 

Dissolve the carmine in the ammonia by means of 
gentle heat, add the water, and filter. 



144 METHODS OF MICROSCOPICAL RESEARCH. 

5. "Wash thoroughly in water. 

6. Place in alcohol for ten minutes. 

7. Clear in xylol and phenol, mount in xylol 
balsam. 

Picro Carmine, Double Staining. 

Picro Carmine as a selective double stain cannot 
be surpassed. The process is as follows : Take of 
carmine 2 grains, liquor ammonia ^ drachm, dis- 
tilled water 1 ounce ; dissolve the carmine in the 
ammonia by means of gentle heat, add the water. 
Dissolve 8 grains of picric acid in 1 ounce of alcohol, 
also by means of gentle heat, and mix the two 
solutions. 

Place the sections in alcohol for one hour; im- 
merse them in the recently filtered staining solution 
for from half an hour to three hours, i.e., until they 
are sufficiently stained ; wash them in alcohol, 
immerse them in an alcoholic solution of picrate of 
ammonia for one hour, and for a second hour in a 
like solution ; in other words, change the solution 
once during the two hours. Place them in alcohol 
for a few minutes, clear in xylol and phenol as 
already described, and mount in xylol balsam. 

Unstained vegetable specimens may be mounted 
in balsam, glycerine jelly, or Farrant's medium. 

The Preparation of Diatomacese. 

Diatoms may be said to be absolutely ubiquitous ; 
recent and fossil, living or dead, they are readily 
obtainable in vast numbers, and of infinite variety. 
In nearly all waters, fresh or salt, still or running, 



ON MOUNTING. 145 

in marshy districts, on the surface of the ocean, or 
attached to algae and shells, they abound in a living 
and growing state, whilst their siliceous valves, after 
the death of the cell-contents, are to be found, in a 
more or less perfect condition, as deposits at the 
bottom of rivers and ponds and on the sea floor. 
From the stomachs of holothurians, ascidians, mol- 
luscs, and of various shell-fish, exquisite varieties 
have been obtained in profusion. As fossils they 
form beds of enormous extent, intermixed with the 
siliceous casts of polycystina, radiolarians, &c., and 
the calcareous shells of foraminifera, whilst fossil 
deposits purely diatomaceous and yielding the most 
beautiful, rare and varied forms have been dis- 
covered in all parts of our globe. In order to clean 
diatoms and render them fit for mounting, as either 
" selected " or " strewed " slides, considerable ex- 
perience and great care are necessary. The sub- 
siliceous varieties especially require very delicate 
treatment, whilst with many of the fossil and recent 
forms, which are intensely siliceous, altogether 
" heroic " and drastic measures must be resorted to 
in order to ensure perfect cleansing of the surfaces 
of the valves. There have been nearly as many 
nostra propounded and published for the cleaning 
and mounting of diatoms as there are genera and 
species of the diatomaceas themselves. Years of 
experience and constant experiments have led the 
author to the conclusion that the simplest and 
safest modes of treatment are the best. For living 
diatoms and especially for the sub-siliceous forms 
(e.g., pleurosigma) the following method will give 
perfect results : Remove all dirt, debris, and salt, 
by repeated washings, thoroughly shaking up the 
10 



146 METHODS OF MICROSCOPICAL RESEARCH. 

gathering each time and allowing the diatoms to 
settle before pouring off the water ; in this way all 
soluble impurities can be removed. When the 
water remains clear, after shaking, pour it off, 
leaving the diatoms as nearly as possible dry ; cover 
them to about one inch in depth with absolute 
alcohol, or the strongest ordinary alcohol, which will 
gradually dissolve and extract all the endochrome 
from the valves ; change the alcohol daily until it 
ceases to be tinged with green. Wash away all 
trace of alcohol, pour off all water, place the diatoms 
in a platinum capsule and heat them to a dull red 
over a "Bunsen" burner; this will separate the 
frustules into single valves, and complete the clean- 
ing of the diatoms, which may be now bottled up 
in distilled water for future mounting. 

To clean Diatoms growing upon Algse or 
Shells. Place the algas or shell debris in a large 
basin (in order to allow space for effervescence), 
cover them with water, add gradually, hydrochloric 
acid, and stir until violent effervescence results ; 
add acid little by little, until effervescence ceases 
thoroughly stirring from time to time strain 
through net of sufficiently fine texture to retain the 
debris whilst the diatoms pass through it. When 
the forms shall have thoroughly settled down, pour 
off all water and place the strained deposit in a 
large test-tube. Boil in pure hydrochloric acid for 
twenty minutes ; add pure nitric acid, and boil again 
for twenty minutes. Whilst boiling add crystals of 
chlorate of potash until complete bleaching results ; 
remove all trace of acids and alkali by repeated 
washings, and examine the forms ; if perfectly clean 
bottle them up for mounting; if, as is sometimes 



ON MOUNTING. 147 

the case, there has been animal matter present, 
which has not been entirely removed, boil in pure 
sulphuric acid for a few minutes, and wash away 
all trace of the acid before placing the diatoms in 
distilled water, for keeping. 

To clean Fossil Diatomaceous Deposits.- 
Sorne fossil deposits are of such extreme hardness 
that it is necessary to resort to caustic potash for 
their disintegration; it is, however, better, when- 
soever possible, to dispense with this powerful 
alkali, since, even in the most careful and practised 
hands, more or less abrasion of the surface of the 
valves will generally be found to result from its 
use. When a fossil deposit will not yield to milder 
treatment, it must be broken into small pieces, 
placed in a test-tube, just covered with the solution 
of caustic-potash, and boiled for half a minute 
(only) over the flame of a " Bunsen " burner ; 
pour off into a glass beaker nearly full of water 
all the deposit which has been disintegrated by 
the potash and repeat the process until all the 
lumps are broken up. Then, when all the forms 
have settled to the bottom of the water in the 
beaker, pour off the water, remove the diatoms into 
a large test-tube, fill the test-tube nearly full ot 
water, add a small quantity of bicarbonate of soda, 
and let the diatoms gently "simmer" in this weak 
solution for two hours. Wash away all soda, and 
boil the diatoms for a few minutes in pure nitric 
acid. Remove all trace of acid by repeated wash- 
ings, and keep the diatoms in distilled water. 

Fossil deposits which can be disintegrated without 
caustic potash should be boiled in a test-tube of 
large capacity, in a moderately strong solution of 



148 METHODS OP MICROSCOPICAL RESEARCH. 

bicarbonate of soda the disintegrated portions 
being, from time to time, poured oft* into a beaker 
and the boiling in bicarbonate of soda repeated 
until all the deposit has broken up, when the 
alkaline solution should be washed away and the 
diatoms boiled for a short time in nitric acid all 
traces of which are to be removed by repeated 
washings and the forms kept in distilled water for 
future use. 

To mount Diatoms " dry." All "dry" 
mounts of diatoms, whether " strewed " or 
" selected," are liable to destruction or deteriora- 
tion from an accumulation of moisture upon the 
under side of the cover, which moisture, sooner 
or later, and in defiance of all precautions, always 
makes its appearance. " Dry mounts " are there- 
fore always more or less unsatisfactory and un- 
reliable and to be avoided as much as possible. 
The best method of mounting diatoms " dry " 
whether for "test" or as "arranged" slides is 
to make a cell of the best asphalt, of the necessary 
thickness, by adding coat upon coat of the asphalt, 
not by making the cell of sufficient depth at one 
operation. Spread tbe diatoms upon the cover, if 
necessary " burn " them, upon the cover, i.e., place the 
cover upon a piece of thick platinum foil and raise 
it, slowly and carefully, to a dull red heat over the 
flame of a " Bunsen " burner; thoroughly heat the 
slip with the asphalt cell upon it ; whilst it is hot 
(and therefore certainly free from all damp or 
moisture) place the, equally hot, cover carefully 
upon the cell, pressing down the cover and making 
sure that it adheres thoroughly and evenly to the 
cell all round run a ring of asphalt round the edge 



ON MOUNTING. 149 

of the cover ; when this is hard, " ring " the cell 
with two coats of white zinc cement, letting the first 
coat dry thoroughly before applying the second. 
To mount Diatoms in Canada Balsam, 

&C. Canada balsam has been, hitherto, universally 
employed as the best medium in which to mount 
diatoms. It has stood the test of time, and proved 
fairly permanent and reliable. For the mounting 
of diatoms, however, hardened balsam re-dissolved 
in benzol or xylol is by no means so satisfactory in 
its results as ordinary balsam, since it does not 
admit of being submitted to the application of direct 
heat, after the slide is mounted ; any attempt so to 
harden the balsam resulting in the production of 
air-bubbles, which carry away the forms in the 
ebullition caused by the endeavour to drive out the 
air, and without heat, applied directly, the balsam 
never " sets " hard. Diatoms should therefore be 
mounted in chemically pure, filtered balsam, diluted 
to the consistency of cream with turpentine. The 
diatoms in " strewed " slides should be allowed to 
fall upon the cover from a " pipette" held at a 
height of three or four inches above it the sudden 
fall of the drop causing the diatoms to spread 
evenly upon the surface of the cover. 1 The drop 
upon the cover should then be dried, very slowly, by 
means of a " Bunsen" burner placed underneath the 
brass table upon which the cover (lying upon a 3 in. 
by 1 in. slip) has been placed. When it is desired to 
mount many slides of the same gathering of diatoms 

1 The even spreading of the diatoms on the cover will be 
further ensured by breathing upon it before allowing the drop 
to fall, whilst in selecting diatoms, if the cover upon which 
they are to be placed is breathed upon, the forms will adhere 
to it. 



150 METHODS OF MICROSCOPICAL EESEARCH. 

the requisite number of covers should be cleaned, 
and three of them laid upon each slip, previously 
breathed upon to cause the covers to rest firmly 
upon it, and the slips laid upon the brass table ; a 
drop of water containing the diatoms should then 
be allowed to fall upon each cover, as previously 
recommended, and the covers can all be dried 
together ; a sufficient quantity of the turpentine- 
balsam should then be put upon the covers, which 
should be placed in a cabinet with trays, or under a 
glass shade, so that all dust may be excluded, for 
twenty-four hours. By this means the absolute 
permeation of the forms by the balsam, and the 
consequent removal of all air are secured before 
the final completion of the mount, an advantage so 
obvious that further comment is unnecessary. The 
covers should then be replaced upon the brass table 
and subjected to gentle heat from a " Bunsen " 
burner until the balsam is hard ; the cover should 
then be taken up by means of forceps, and a slip 
being made hot the cover should be placed precisely 
in the centre of the slip, 1 which should then be held 
over the "Bunsen" burner until the balsam runs 
to a neatly bevelled edge round the cover. No air- 
bubbles will trouble the mounter if this process is 
carefully practised. This is the so-called " exposure- 

1 In order to ensure the placing the cover exactly in the 
centre of the slip, either of the three following devices will 
be found convenient : 

Firstly. Upon a small white tile draw, in china painting 
colours (which should be burnt in), a 3 in. by 1 in. space (repre- 
senting a slip), with its precise centre strongly marked. This 
tile will be found of great use in suddenly cooling balsam, when 
that may be desirable. 

Secondly. Cover a 3 in. by 1 in. slip with white paper, rule 



ON MOUNTING. 151 

process," which was invented by the author in 1870, 
and the success of which induced him to devise the 
necessary modifications upon it and to construct the 
necessary appliances in order to adapt it to the 
mounting of histological and other preparations. 
Slides of " selected " diatoms should be mounted in 
the same manner, the forms being picked up by 
means of a carefully chosen hair from the dried hide 
of a cow the soft and flexible hairs taken from 
the under side of the neck being those most suitable 
for the purpose. These hairs are to be affixed to 
the cedar sticks used with " camel-hair pencils " by 
means of sealing-wax dissolved in alcohol, and a 
number should be prepared, having the hairs of 
different lengths in order to obtain varieties of 
strength and elasticity. Some hairs should be 
mounted of considerable length, as the mounter of 
diatoms and other such minute specimens must 
learn to put them back symmetrically into their 
required positions under the microscope, and in, the 
balsam when they float away ; a little practice will 
soon lead to the acquirement of this absolutely 
necessary skill, and it will be found not difficult to 
heat the hardened balsam (many times if necessary), 



lines faintly from each top corner to its opposite bottom corner ; 
the intersection of these lines will give the exact centre of the 
slip, which can be marked by a dark spot. 

Thirdly. "With a writing-diamond make, upon a slip, placed 
on a turn-table, concentric circles, gin., fin., fin., and -pn. 
in diameter ; each sized cover can then be placed precisely 
in the centre, if the slip on which the cover is to be placed 
is laid upon that upon which the circles have been described, 
the circles, of course, being clearly visible through the super- 
imposed slip. 



152 METHODS OF MICEOSCOPICAL RESEARCH. 

and whilst it is hot to, very rapidly, replace a form 
in its desired position, whilst with a fine needle all 
hairs and other imperfections can be removed and 
absolute cleanliness ensured. 

About a quarter of a century since, when slides 
of "selected" and "grouped" diatoms, &c., were 
in the zenith of their popularity, and finding, from 
painful experience and repeated failures, that the 
successful mounting of these and other such pre- 
parations was surrounded by almost insuperable 
difficulties, in respect both of affixing the siliceous 
valves to the thin covers on which they were to be 
placed, and the selecting perfect forms from the 
debris and refuse of the cleaned diatomaceous 
material, and safely transferring them to the slide 
in course of preparation, the author, in order to 
conquer these difficulties, devised a cement (for 
which the formula will be given in its place), and 
which has proved equally safe, permanent and 
invisible, even under the highest powers of the 
microscope, and, indeed, the only really reliable 
method of fixing diatomaceae, spiculse, polycystina, 
&c. It is the only cement, also, so far as the 
author knows, which has stood the test of many 
years, all other cements used for this purpose (and 
all of which he has tried) having sooner or later, 
and generally sooner rather than later, succumbed 
t o the disintegrating action of the Canada balsam, 
or other media in which the forms have been 
mounted, with the result that they have become 
detached from the cover and valuable and expen- 
sive slides thus destroyed and rendered worthless. 
Finding also by equally annoying experience that 
the so-called " mechanical finger " and other such 




COLE'S DIATOM MOUNTING STAGE. 



ON MOUNTING. 153 

devices for selecting and transferring diatoms, &c., 
to the cover were both cumbersome and uncertain 
in their use and action, and that diatoms when 
picked up by means of a hair, a bristle, or a brush, 
of which the point was coated with gum, or some 
other sticky substance to which they adhered (or 
did not adhere), resulted in constant loss of rare and 
valuable forms, in course of transference to the 
cover, breakage, loss of time, and general uncleanli- 
ness, he invented, for his own use, the " Diatom 
Mounting Stage," of which the accompanying 
diagram is an exact and full-size illustration. 

This stage is, of course, intended to replace the 
ordinary mechanical stage of the microscope, and 
that stage being temporarily removed, the diatom 
stage is firmly fixed to the flat table of the main 
stage by means of two screws passing there- 
through and screwed into the diatom stage. The 
stage must, of course, be so made that the central 
circular orifice shall precisely coincide with that of 
the stage of the microscope with which it is to 
be used. Its description and the manner of its 
employment are as follows : A A is a flat brass 
plate with a central circular orifice for the passage 
of reflected light from the mirror of the microscope. 
B B are grooved side-pieces, in which slides, very 
smoothly and evenly, the frame C. This frame is 
retained in position at any point in its upward or 
downward movement by two springs D D. It has 
also two smaller springs E E, the use of which will 
be described presently. F is a slightly raised bar 
ending in a handle G. H is the raised upper rim 
of the stage through which a screw passes, I. J J 
are the raised sides of the upper portion of the 



154 METHODS OP MICEOSCOPICAL RESEARCH. 

stage, and K is a piece of glass (its termination 
being represented by a dotted line) which slides 
into grooves in the sides, J J, and which is intended 
to secure, and practically does secure, absolute 
cleanliness to the slide during its preparation. 

Now, the stage being fixed in its place by its two 
screws, an ordinary glass slip (3 in. by 1 in.) is taken, 
and by means of a turn-table a very minute circle 
is marked, with a writing diamond, precisely in its 
centre, upon this circle is placed the smallest 
possible drop of distilled water, and upon this drop 
of water the cover, on which the diatoms are to be 
mounted, is placed, and of course firmly held in 
its position by capillary attraction ; care must be 
taken that the circle on the slip shall exactly coin- 
cide with the centre of the cover. The slip is then 
to be placed under the springs E E, and the stage 
must be so fitted to the microscope that when the 
rim F of the sliding portion of the stage is drawn 
down by the handle Gr it is stopped by a pin L 
when the small circle under the cover on the slip 
is precisely in the centre of the field of the micro- 
scope. The slip being firmly held in its position 
by the springs E E the absolute centrality of the 
"mount" is ensured, if care be taken that the 
cover is not moved. Now, having spread and 
dried some diatoms upon another slip, place the 
slip upon the lower portion of the frame C C. This 
slip can be freely moved, and if the screw I is 
screwed out to its extreme length it is obvious that 
if the frame C C is raised to the lower end of the 
screw the lower edge of the movable slip can be 
passed across the field of the microscope and any 
desired diatoms selected from it. After removing 



ON MOUNTING. 155 

the diatoms from that line the screw is to be 
screwed down a little so as to prevent the frame 
sliding up so high, and this gives another line of 
diatoms for selection, and this can be repeated until 
the entire slip has been thoroughly explored. In 
this manner nothing can be overlooked, since the 
action of the screw ensures the return of the slip 
to precisely the same spot. Now, the cover on 
which the diatoms are to be mounted is to be pre- 
viously prepared by placing upon it a small drop 
of the cement already referred to, which cement 
has to be kept moist during the selecting and 
arranging the diatoms. For this purpose an 
ordinary glass tube with a bulb is to be procured 
and mounted in a piece of brass tubing, having a 
ball and socket joint, and which is to be made to 
fit into that part of the microscope stand which is 
occupied by the side reflector when in use. To 
the shorter end of the glass tube a piece of india- 
rubber tubing is to be attached, and a small piece 
of glass tubing, to be used as a mouthpiece, is to be 
inserted into the other end of the elastic tubing. 

Now this " breathing apparatus " being fixed in 
its place on the stand of the microscope, the longer 
end of the glass tube is to be placed about three 
inches from, and level with, the thin cover on the 
glass slip, and the frame C C drawn down to the 
pin L ; now breathe (do not How so as to blow away 
the diatoms) upon the cover, and it will be found 
that the moisture from the breath will at once 
liquefy the cement, and with a bristle mounted on 
the cedar stick of a camel-hair pencil as already 
described carefully remove all dirt, if any, from 
the cover, then from the lower slip select a diatom 



156 METHODS OF MICROSCOPICAL RESEARCH. 

and if necessary, wash it on the slip in the moisture 
from the breath, which moisture will also cause the 
diatom to adhere to the bristle ; now draw down 
the sliding frame and place the diatom on the cover. 
In this way any number of diatoms, &c., may be 
transferred from the lower slip to the thin cover 
and arranged as desired, and when the moisture 
from the breath has evaporated, it will be found 
that the forms are firmly affixed to the cover, whilst 
the glass roof over the " mount " has preserved it 
from all danger or dust during its preparation. 
The cement with which the diatoms are to be 
affixed to the thin cover is made thus : In 1 
ounce of distilled water dissolve 2 grains of best 
gum arabic, without heat. When the gum is dis- 
solved add 3 minims of glacial acetic acid, to pre- 
vent fungoid growth, and the least trace of sugar 
to prevent the solution from cracking as it dries. 1 
The solution should be carefully filtered through 
the finest filter paper, and the filtering repeated at 
intervals of a few weeks. 

For years experiments were in progress in the 
hope of discovering a medium in which to mount 
diatoms, which should be more satisfactory, in 
respect of its refractive index, than Canada balsam, 
and equally reliable and permanent ; many such 
media have been tried phosphorus, balsam of 
tolu, oil of cloves, and others ; but it is unneces- 
sary to enter into details concerning them, since the 

1 For affixing delicate insect dissections to the slip or cover, 
this cement should be made with 8 grains of gum arabic 
to the ounce of distilled water, and for polycystina or forami- 
nifera with 10 grains, and ior sponge spiculae with 6 grains to 
the ounce. 



ON MOUNTING. 157 

desired results "have been obtained by Dr. Yan 
Heurck, who succeeded in providing an admirable 
substitute for balsam in gum-styrax. It will pro- 
bably be found that diatoms mounted in gum-styrax 
are less liable to accidents than balsam " mounts," 
as the latter becomes resinous in time, and the 
covers are liable to " spring," the result of which 
is the appearance of prismatic colours in the balsam, 
which are not only a great eyesore, but sadly 
deteriorate the slide. Grum-styrax, the method of 
preparing which for use has been described at page 
105, may be considered absolutely permanent and 
unalterable. The solution is used in the same 
manner as Canada balsam. Styrax solution is even 
easier to work with than balsam, and air-bubbles 
are not produced in it by the application of heat. 
Monobromide of naphthaline as a medium in which 
to mount diatoms, and the method of its application, 
have been described at page 105. 

Cleaning and Mounting Polycystina and 
Radiolaria. 

The siliceous shells of these lovely organisms, 
the beauty of which is only equalled by their variety, 
are found in profusion, and intermixed with diatom- 
aceous valves, often of extreme beauty and rarity, 
in the deposits (or so-called " earths ") of Barbados, 
the Bermudas and the Nicobar Islands, whilst an 
entirely new and magnificent deposit was discovered 
by the late Captain Perry, of Liverpool, at Jeremie, 
Haiti, which contains not only a large number of 
novel species of polycystina, but many entirely new 
varieties of diatoms. The celebrated " Challenger " 



158 METHODS OF MICROSCOPICAL EESEAECH. 

dredgings also gave to science innumerable and 
splendid varieties. 

The polycystinous " earth " should be broken 
into small pieces, about the size of a nut, and boiled 
for half an hour to an hour in a strong solution of 
common " washing soda," the disintegrated por- 
tions being poured off into a large vessel containing 
clean water, from time to time, and the boiling in 
soda repeated, as also the pouring off, until the 
whole mass is perfectly broken up. When the 
disintegrated matter in the large vessel shall have 
thoroughly settled down it should be subjected to 
several washings, in order to remove the soda, the 
material being allowed to " settle" thoroughly after 
each washing. It should then be removed to a 
beaker or wide-mouthed bottle which should be 
filled up with water, and after being thoroughly 
stirred or shaken up, the material should be allowed 
to settle for thirty seconds only, in a depth of 6 
inches of water, and the supernatant fluid and its 
floating particles poured off into a large vessel ; 
this process should be repeated three or four times, 
and will give the heaviest density of sand and 
polycystina. Repeat this process with the matter 
in the large vessel, allowing it to settle for two and 
a-half to three minutes, and the density containing 
the small polycystina will be obtained. Subject 
the remaining matter to like treatment, allowing it 
to settle in 6 inches of water for twenty minutes, 
and the density consisting of the debris of polycy- 
stina and of diatoms will result. Now boil each 
separate sediment in nitric acid for fifteen to twenty 
minutes, remove all trace of acid by repeated wash- 
ings, and finally boil each density in a weak solution 



ON MOUNTING. 159 

of bicarbonate of soda in a test-tube of large 
capacity for an hour ; this will remove all flocculent 
matter, and after repeated washings, perfectly 
clean polycystina will be obtained. The heaviest 
density consisting of sand and the largest poly- 
cystina should be placed in a test-tube with about 
3 inches of water and subjected to rotatory motion, 
this will cause the polycystinous shells to rise above, 
and to free themselves from, the sand, and they 
can be poured off into a wide-mouthed bottle or 
beaker, the sand being left at the bottom of the 
test-tube. This latter process should be carefully 
conducted and repeated several times in order that 
no large and perfect shells may be left amongst the 
sand. Each density should then be bottled in 
distilled water. Polycystina may be mounted in 
balsam in the same manner as diatoms or "dry." 
Beautiful slides can be produced by calcining the 
shells upon a piece of thick platinum foil, or in 
a small platinum capsule, and mounting them 
"opaque." The neatest and best method of pre- 
paring such slides is to make a disc half an inch in 
diameter in the centre of a slip, allowing it to 
harden for some days. A half-inch cover is then 
to be cleaned and a sufficient quantity of balsam, 
thinned with turpentine, put upon it; the polycy- 
stina are then to be placed in the balsam in suffi- 
cient quantity to form a surface over the cover 
when evenly spread upon it ; the cover with the 
polycystina is then to be put aside (as recommended 
with diatoms) for twelve or twenty-four hours, in 
order that the balsam may thoroughly permeate 
the forms and all air escape ; the balsam is then 
to be hardened by gentle heat, as already described, 



160 METHODS OF MICROSCOPICAL EESEARCH. 

and the cover is to be laid upon a slip, with the 
balsam upwards, and held over the flame of a 
Bunsen burner until the balsam is liquefied; the 
polycystina are then to be evenly spread upon the 
cover by means of a needle ; when the balsam is 
cold a small drop of benzol-balsam is to be placed 
upon its surface and a clean half -inch cover to be 
warmed and carefully lowered upon the balsam. 
The polycystina will thus be mounted between two 
covers; now turn the "mount" over, so that the 
under surface of the lower cover shall be upwards, 
and coat this with asphalt, put the mount aside for 
a day or two, in order to allow the asphalt to be- 
come thoroughly hard, then having made in the 
centre of a slip a disc of asphalt of the same 
diameter as the "mount," and allowed that also to 
dry thoroughly, put upon this disc a small drop of 
(cold) " French liquid glue," 1 place upon this the 
asphalted under surface of the " mount," and when 
the glue is quite dry finish off the slide with either 
asphalt or white zinc cement. 



1 This is to be obtained from any chemist, and is a most 
admirable, reliable, and cleanly cement. 



161 






CHAPTER VII. 



On Microscopical Drawing and Painting. 

A GREAT teacher has said " Drawing should be 
considered not an accomplishment, but a necessity. 
Learning to draw is learning the grammar of a 
language. Anybody can learn the grammar, but 
whether you have anything to say is another 
matter." To the naturalist this accomplishment is 
of great importance ; accurate illustration adds to 
the value of written description. At every point 
the microscopist is sensible of its deeper signi- 
ficance. Such a control quickens the perception, 
excites exact observation, and creates an interest 
beyond research, admiration, or curiosity. The 
compactness of the vision presented by the micro- 
scope so rivets the attention, that changes, dis- 
closures, development of activities, in organisms 
often lost and swept away, after cursory examina- 
tion, rouse in a zealous observer an impatient desire 
to possess some power, beyond words, to place on 
paper a memorandum or record, however rough, of 
things rarely discovered under the same conditions. 
This ability is a result of practice. There is no 
11 



162 METHODS OF MICROSCOPICAL* BESEARCH. 

royal road but that traversed by enthusiasm and 
earnestness. Sketches from the hands of a dex- 
terous microscopist, marking first impressions, are 
often more valuable than, and superior to, the formal 
work of the mere draughtsman, who may not even 
know the significance of the subject, especially when 
the result is a replication of drawings made by the 
actual 'observer. He necessarily falls into one or 
other of two errors ; he mends and improves, or 
obscures material points by drifting into formal 
monotony ; a microscopical draughtsman must 
essentially be a microscopist, and work direct from 
actual observation, completely understanding the 
matter before him. 

There are three well defined characteristics of 
microscopical representations, drawings of tissues, 
or minute organisms, requiring for elucidation high 
powers, delicate conditions of light, conducted 
under careful observation and technical skill, satis- 
fying the highest biological research, in its progress 
demanding rigorous precision ; then, rapid sketch- 
ing, catching features, graphic memoranda ; without 
hesitation, or the assistance of the camera-lucida 
jotting down, and washing in, with tints, un- 
expected appearances, this readiness should be 
cultivated by those desirous of adding record to 
observation; many most important phases in the 
sequence of activities have been seen and passed 
over, when a few rough lines would have induced 
and helped further research, but beyond this tenta- 
tive work, and the stern formality of scientific 
requirement, is a finished " picture ; " at this 
crucial point the capability of the microscopist and 
artist blend, involving knowledge of the subject, 



ON MICROSCOPICAL DRAWING AND PAINTING. 163 

the arrangement of optical apparatus, judgment, 
and study in the methods of procedure. A drawing 
may be true in its scientific aspect, and possess 
artistic features of decided interest the one may 
incorporate the other. The illustrations of Mr. 
G-osse's books are instances of this peculiar quality. 
" Still Life " has arrested the attention of artists 
of all time, from Missal Illuminators to Royal 
Academicians ; such results have no scientific im- 
port, but like all art products, awaken gratification 
in appreciating the power applied in producing 
their essence, and without degrading the legitimate 
functions of the microscope, it is possible to extract 
from its revelations, models of exceptional charm 
and excellence, associated, moreover, with scientific 
value. Although the bias of an expert microscopist 
and practised artist may not often touch the same 
mind, it is certain that when a keen perception is 
directed to complications of beauty, with rare con- 
ditions of light and effulgence of colour, the in- 
strument becomes the very touchstone of artistic 
feeling, and beyond mere beauty (which, in visible 
nature, is inexhaustible) there are revelations of 
structural form, quaint elegancies, mysterious 
changes of tissues, and embryological developments, 
under radiances, hidden, not only from ordinary 
familiarity, but even from the cognisance of many 
w4io have not had the opportunity of exhausting 
the resources of a fine instrument, with all its 
accessories. It may be urged that few have the 
ability to approach art of this description, but the 
power of drawing quickly develops itself, especially 
when stimulated by special and eager interest, con- 
centrated on special objects ; no one led by inelina- 



164 METHODS OF MICROSCOPICAL RESEARCH. 

tion to contemplate what may be seen under such 
circumstances, can be destitute of an appreciation 
of art in its most exalted sense. The education of 
the eye (the basis of aesthetic culture) as exercised 
by the fascination and mental excitement of micro- 
scopical research, progresses in a degree hardly 
yet understood or appreciated ; every student has 
within his mastery this power ; the manipulation of 
the instrument, the means of display, the use of 
materials, are matters of expertness, and are ex- 
tensively self-taught; possibly some instruction may 
do more than groping alone, but in the end expe- 
rience is the best master. It is proposed in this 
essay to detail such an actual experience in words, 
directed more to mental judgment than technical 
education. 

A microscopical drawing may be absolutely true, 
and an artistic grace secured, by preserving line 
for line what is actually presented, assuming the 
preparation to be fairly perfect ; in other words, 
not drifting into a stilted diagrammatic style, or 
wandering from close observation, because the sub- 
ject appears to have a certain regularity ; no two 
cells, vessels, or fibres are absolutely alike ; to give 
" life " to a picture, every part of the structure 
should be a portrait, the pencil deviating from 
accuracy melts into falsity and confusion, uniformity 
is fatal, and obscures important differentiation of 
parts ; again, in order to delineate what is expected, 
or wished to be seen, aiming at " correction" is to 
be avoided ; it is better to draw imperfections, if 
they be present, an overlapping or torn structure 
often reveals an important fact, so patent is this, 
that a " fabricated " drawing may be detected in a 



ON MICROSCOPICAL DRAWING AND PAINTING. 165 

moment, especially of diatomaceous or infusorial 
forms a broken fragment, a solitary individual 
is the clue to a perfect whole, or group ; such built 
up arrangements have no charm beyond technicality. 
A good representation possesses a mingled quality 
of accuracy and imperfection, a paradox, which 
stamps its value ! Suppose a preparation of vertical 
section of human scalp of rare excellence, double 
stained, disclosing beauty in many perfect hairs 
traceable in their course direct from the base of 
the bulb, embedded in the follicle, and emerging 
from the cuticle above. In cutting a section of 
such delicacy it would be impossible to avoid slicing 
through a hair or two diagonally, thus leaving the 
tops of some, the ends of others; this result or 
defect is a feature of significant interest from an 
art point, faithfully copied it gives life and cha- 
racter. In a diagram, the imperfection, by com- 
parison with perfect hairs, might be remedied, the 
mutilated parts " restored ;" but such an interfer- 
ence destroys at once the graphic quality of the 
picture, adding nothing to its scientific interest. 
Absolute accuracy in depicting what is presented 
may, however, in some cases, be qualified, and truth 
evolved by a knowledge of the structure as it should 
appear, particularly in cellular tissues, in close con- 
tact. In such cases the artist ought to be cognisant 
of elementary forms, as arranged under contiguous 
pressures, and the position of spherical, oblong, or 
cubical elastic cells, as affected by juxtaposition in, 
over, or under spreading layers. Coupled with the 
perspective of such conditions, this facilitates pro- 
gress. In opaque subjects, under binocular vision, 
where the rotundity of a reticulated surface fades 



166 METHODS OF MICROSCOPICAL RESEARCH. 

in dimensions, and shadow, in different lines, this 
abstract knowledge is important, and should be 
acquired, as many objects could not be effectively 
represented without its study always keeping to 
general appearances ; it is an ability which removes 
difficulty in unravelling the disposition of parts, 
especially under high powers ; when sections are 
cut either too thin, slightly oblique, or disrupted 
by the knife, the mechanical interferences of parts, 
when understood, may be restored. The functions 
of an artist, cognisant of a condition of antecedents 
may be fairly exercised in the progress of a draw- 
ing, but it must never trench upon absolute truth 
and discrimination in treatment a drawing may 
be ruined in a moment by a false line involving 
impossibility of structure ; to a critical eye, this 
is fatal. In fine work, dealing with malpositions, 
shrinkage of tissues, disseverances and pseudo- 
appearances inevitable even in the finest prepara- 
tionsthe utmost judgment is required. 

The effect of a microscopical drawing is enhanced 
by its inclusion in a circle surrounded by a black 
margin forming a square. The size of the circle 
is important it may be too large, or too small ; 
experiment proves that a space three inches and 
three-quarters in diameter approximates nearest to 
the impression made on the mind of a " field " as 
seen with a B eye-piece ; this circle may encompass 
magnifications under any power. A metal plate 
four inches and three-quarters square, with an 
opening of the dimensions given, should be pro- 
cured, this ascertained gauge will soon prove a 
necessity ; placed on a drawing block, a pencil 
swept round the circle and outside the square gives 



ON MICROSCOPICAL DRAWING AND PAINTING. 167 

the interior for the drawing and the lines for back- 
ing with indian ink these discs should be prepared 
before the work is commenced, and the importance 
of this arrangement will be shown hereafter. 

The help derived from the caraera-lucida is strictly 
limited, it cannot be employed beyond a certain 
point, no elaboration can be effected by its pro- 
longed use, it should be discarded the moment its 
legitimate purpose of marking points and positions 
is achieved ; those experienced in its employment 
always feel a sensible physical relief, and " breathe 
again " when it is set aside to settle down to the 
earnest work of direct vision. The application of 
the camera-lucida to the instrument is sufficiently 
familiar ; the microscope, if a binocular instrument, 
having been rendered monocular by withdrawing 
the prism, the camera-lucida is slipped over the A 
eye-piece which should always be used (higher 
eye-pieces expand the field beyond the fair range 
of the instrument). The object being clamped, the 
microscope is depressed into a true horizontal posi- 
tion (if not, a distorted picture would result), and 
the lights adjusted ; the distance from the object to 
the eye-piece should be nearly equivalent (if any- 
thing a little more) to the reach from the eye-piece 
to the paper. With a microscope standing ten or 
twelve inches high (a Ross No. 1) this condition 
would reveal the phantom of the object, outside or 
about filling a circle of the dimensions given on the 
drawing block ; if any difference of over, or within, 
lapping appear, it may be remedied by raising or 
otherwise adjusting either the paper or the micro- 
scope so as to obtain a perfect coincidence of the 
vision and the circle ; the importance of a measured 



168 METHODS OF MICROSCOPICAL RESEARCH. 

disc is now manifest, proportion is affected by dis- 
tance from the eye-piece, and with this gauge and a 
stage micrometer, a drawing may be kept within 
measurable bounds ; difficulties have existed as to 
amplifications expanded by the camera-lucida; ab- 
solute accuracy may be ascertained by the use of 
micrometers as far as the eye-piece is concerned, 
but the known diameter of a circle on which the 
image is projected is an easy factor in such cal- 
culations, beyond this, the circle is mechanically 
useful, as, if the block should slip when using the 
camera-lucida, there is an ascertained line for re- 
adjustment. The light on the paper should be in 
excess of that from the object ; speed and precision 
are essential, quickly make recognised points and 
lines, never attempt to draw detail, nothing fatigues 
the eye or distracts the mind more than the pro- 
longed employment of the camera-lucida. No ad- 
advance can be made by its continued use, any 
attempt at elaborate work ends in confusion. Cul- 
tivate the " knack "of seeing at the same time, and 
in the same position, the reflections and the image 
of the tracing point (the hardest pencil, sharply 
cut) ; it is not necessary to strain the sight to keep 
the entire field always in view, there is a condition 
of steady gazing, the eye not too close to the prism, 
when PARTS of the object can be taken separately, 
but this is a result of the facile use of the instru- 
ment. The neutral tint glass or any form of reflec- 
tor, giving only one, and consequently, a reversed 
image, is useless for afterwards continuing a draw- 
ing from direct vision, but with any description of 
camera-lucida, the pencil, once placed on the paper, 
should not, if possible, be lifted until all determining 



ON MICROSCOPICAL DEAWING AND PAINTING. 169 

lines are fixed ; the eye (unsteady at the best) and 
the pencil point must be in unison. Keeping the 
pencil on the paper preserves " the place." 

In arranging any object for drawing, it should be 
sufficiently magnified to show everything bearing 
upon its elucidation, and, as a rule, an isolated 
subject, a complete form, ought to occupy, as 
nearly as possible, the entire field. Some specimens 
necessarily overflow the circle surfaces of injected 
preparations, botanical sections exhibiting features 
requiring the highest magnification consistent with 
the preservation of a focal plane which, obviously, 
cannot be fairly disclosed (except at a loss of im- 
portant detail), are beyond the scope of the circle 
of popular survey. It must then be abandoned, 
the drawing spread out, and made in sections by 
shifting and combining visions. Using the camera- 
lucida any part may be carefully drawn, making 
two or three (if angular, the better) prominent 
points, corresponding with similar appearances in 
the subject. These marks or tri-angulations (as 
near the margin of the field as possible), must be 
remembered, the position of the object is then 
moved by stage adjustments, and another part of 
the field arranged, the included marked points are 
coincided, by shifting the block of paper, and further 
outlines expanded ; in this way the camera-lucida 
may be used under high powers with four or even 
six combinations of vision and the parts, with 
care, will " read into " each other, and result in 
a drawing of considerable dimensions, perfectly 
mapped, and true in contour ; it may then be con- 
tinued part by part. 

After faint outlines and points of certainty are 



170 METHODS OP MICROSCOPIC A.L RESEARCH. 

securely indicated, the microscope is placed in posi- 
tion, and with B eye-pieces drawing from direct 
observation commenced ; prolonged work is facili- 
tated by removing the caps of the eye-pieces ; when 
attention is continually diverted from the instrument 
to the pencil, the fatigue is lessened by keeping 
the eyes some distance from the glasses, cultivating 
a faculty of losing the recognition of the entire 
objects, only directing the alertness of vision to the 
particular part under consideration ; in fact it is not 
necessary, nor is it prudent, to strain the sight to 
keep the full blazon of the field under observation, 
and this rule may, with advantage, be applied to 
the general use of the microscope. At this point, 
steady work commences, faint camera indications 
are studied, lines corrected and strengthened, either 
with pencil, or better, a fine sable brush or pen, 
carrying a mere tint of indian ink or " Payne's 
Grey." Extreme care is necessary ; no mistake of 
line can be permitted ; paper, intended for such 
drawing, and delicate after colouring, does not 
permit erasure, or the contact of any rubbing out 
substance ; and consistent with the subject, too 
much fine line cannot be put into the work ; no 
attempt at shading, either with pen or pencil, must 
be attempted. The lines being perfected, and the 
subject, as it were, " modelled," the painting may 
now be cautiously commenced. The absorbent 
quality of paper (well known to those accustomed 
to water-colour sketching), interferes with, and 
sometimes assists in securing artistic results ; with- 
out entering into the rationale, but bearing upon 
the point, it may be mentioned that no wash or 
even line should be superimposed upon or carried 



ON MICEOSCOPICAL DRAWING AND PAINTING. 171 

over another until the first be perfectly dry ; stip- 
pling should show a granulated appearance, lost 
when touches are allowed to run into and become 
absorbed by each other. 

Illumination, and its diversities for art work, are 
of as much importance as the amplification. Power 
and light should be adapted to each other, and 
to the character of the subject, its mode of prepara- 
tion, and what it is expected to reveal. The light, 
whether from gas (argand burner), or oil, should be 
capable (in the case of gas, by means of flexible 
tubing,) of being placed in every possible position, 
from the surface of the table to, or even above, the 
level of the stage. Ordinary transparent objects, 
under low powers, are sufficiently shown with 
transmitted light from the mirror, modified through 
a diaphragm of waxed tissue paper ; ordinary pre- 
parations of insects cannot be better displayed ; the 
best reflected light is from the side speculum, col- 
lected from a flame through an intervening plano- 
convex lens, on a separate stand. In all observations, 
even the simplest, accuracy of liglit-focus (often 
neglected) is important. For powers beyond the 
half -inch, transmitted light is aided and improved 
by the purity and control afforded by the achromatic 
condenser, an instrument in the hands of novices, 
not always well managed, or sufficiently appre- 
ciated ; focussing on the same plane as the object, 
the source of light, it is capable of regulating 
intensity, purity, and deviation of rays by aper- 
tures and stops, with which it is supplied and thus 
the most varied combinations may be secured. Its 
use should be thoroughly mastered, as it produces 
the most beautiful, instructive and even amusing 



172 METHODS OF MICROSCOPICAL EESEARCH. 

effects. For instance, with a half-inch objective 
and full aperture, carefully focus, on a ground glass 
slip, the flame of the lamp, now interpose a dark 
stop which should occupy, in the centre, about one- 
third of the field ; removing the slip, replace ifc 
with a group of (say) volvox-globator ; the plants 
will be seen rolling from the outer ring of pure 
transmitted light into the central black disc, where 
they appear like emeralds; free-swimming rotifers 
will pass backward and forward, from the outer 
ring of parallel rays, into the eclipse of the dark 
stop, where they become by oblique radiations, self- 
luminous ; no finer example as showing in one 
field, at the same moment, two extremes of illumina- 
tion could be placed before the microscopical artist, 
or an ordinary observer, to prove what may be 
effected by an adept in the use of this beautiful in- 
strument. With low powers striking presentations 
of artistic illumination are under easy control; in 
particular, the use of the paraboloid combined with 
light from above. An experienced microscopist 
is familiar with all these methods but the artist, 
alert and eager for experimental conditions, often 
hits upon effects not generally applied, possibly sac- 
rificing scientific truth to aesthetic desire ; a result of 
positions, and foci of illuminators their accurate 
or eccentric adjustment, cutting off central or 
peripheral rays, dispersing or half obscuring light 
by intervening transparencies. The importance of 
such combinations is paramount in the examination 
of semi-opaque objects immersed in a thick bed of 
medium, without pressure. These preparations are 
in parts dense, even solid, combined with tissues of 
extreme delicacy and transparency ; nothing being 



ON MICKOSCOPICAL DRAWING AND PAINTING. 173 

crushed, they shew the impossibility of revealing 
the correlation, or association of parts, by mere 
transmitted light or in any way, except (may it be 
said) by artistic discernment. The head, and sur- 
rounding parts of an insect, prepared in this way, 
with pure light from beneath and above, discloses a 
combination of form and colour of surpassing 
beauty; the blaze from the speculum sweeps over 
the opaque parts with reflections revealing the most 
exquisite tints, while the paraboloid shows, in actual 
perspective, the parts beneath in all their natural 
colour, and bathed in refulgence. An opaque 
polypodom, touched by such reflections, while the 
extended polyps are illuminated from below, is 
another instance, amongst many, of beauty, exalted 
by light. 

For purely opaque objects, the only good light is 
from the speculum, by no other means can the 
finest effects of colour and shadow be obtained. It 
should be fitted to tho stand of the instrument, not 
to the stage, nor should it slip on the front of the 
objective. On the stand it can be moved without 
disturbing the object or the focus. The old fashioned 
Lieberkuhn cannot be used ; it requires an object 
to be prepared in a particular way, and as an 
illuminator is palpably defective; the light com- 
pletely surrounds and enwraps the object ; brilliancy 
is present, but no contrasts. In using reflectors, 
the lamp should be placed close to the level of the 
stage, within easy reach of condenser and speculum. 

In painting purely opaque objects under top light 
the treatment of background deserves attention ; eggs 
of insects or parasites are generally attached to frag- 
ments of wood, leaves, cuticles, hair or feathers, it 



174 METHODS OP MICROSCOPICAL RESEARCH. 

enhances the effect and beauty of a representation, 
if such details are carefully painted, and the rest of 
the field delicately stippled up with indian ink, to 
the edge of the circle. This applies to many sub- 
jects threads of algse, or vegetable stems support- 
ing such objects as fixed rotifera, polyzoa, &c., 
introduced into a drawing, add greatly to the 
interest and make most attractive pictures. Any 
prepared mount or specimen should be as perfect as 
possible, and considerable experience is necessary in 
order to decide what is fairly good as a prepara- 
tion and ivorth drawing. Common objects of 
easy procurement from the woods, the garden, and 
the stream, are exquisite models for the draughts- 
man, their excellence, interest, and freshness are 
necessarily superior to even the admirable results 
now obtained by professional preparers, aided by 
mechanical appliances, and rare skill in the use of 
reagents and staining fluids. 

It is obvious that objects under polarised light, 
are practically beyond the power of faithful delinea- 
tion ; in all painting, whether in local tint or 
shadow, purity of colour and the preservation of 
brilliancy is of the first importance ; in order to 
render, beyond a mere semblance, any subject under 
the polariscope, it would be necessary, if such a 
power were possible, to dip the pencil into light 
itself, and an insuperable difficulty exists in the per- 
manent preservation of the adjustments necessary 
for future work, the slightest touch, or . alteration 
of any part of the instruments, and even an obscure 
change, beyond all control, in the quality of the 
source of light alters the entire gamut and con- 
sonance of colour, impossible to re-establish, yet, 



ON MICROSCOPICAL DRAWING AND PAINTING. 175 

if selenite films be dispensed with, some results may, 
with care, be recorded ; petrological preparations, 
the dichroism of crystals, sections of shell, bone, 
scales, horn, and other semi-transparent organic 
structures of varying densities, reveal points of 
interest only seen under such conditions and may 
be noted ; but considering that the most exalted 
light at the command of the artist, is the white 
of the paper (in all cases, to be jealously preserved), 
and that the polariscope discloses the purest coloured 
lights, associated with complementary tones of every 
gradation, it is clear how futile are the resources of 
the palette to depict the lustres and unisons of 
tones as revealed by this fascinating instrument. 

Structure and its thoughtful exposition is the 
limit of draughtsmanship, and it is here that the 
photographic lens as a delineator fails; the superiority 
of work produced by a hand guided by cultivated 
observation as compared with a photograph is the 
operation of a mind capable of expressing combined 
and superimposed tissues, in having at command a 
control and adjustment of various planes of surfaces, 
and without militating against scientific truth, seek- 
ing for, and obtaining even picturesque effects, 
this important power is felt when searching the 
depths of an opaque injection, or peering into 
intricacies of tissues. Tbe objective used in micro- 
photography, especially if it be a high power (un- 
like the penetrating quality of an ordinary portrait 
lens), is strictly limited to one, and that a very 
delicate focal plane requiring a fine and FINAL 
adjustment, enhanced by the difficulty that the 
visual and chemical foci of microscopic objectives 
do not coincide, entailing a manipulation which 



176 METHODS OP MICROSCOPICAL RESEARCH. 

never touches perfect precision ; on the other hand, 
a draughtsman may arrange a minute and just 
perspective of parts, absent in a photograph, antici- 
pating the presence of relative parts, and having 
at command the fine adjustment, he can feel his 
way, conscious that at the slightest touch a fresh 
point, perhaps an important revelation, flashes into 
sight, supplying a link to the better understanding 
of the whole. A drawing, produced under thought- 
ful guidance, conveys to an appreciative observer 
an attraction totally absent from a photograph; 
the latter may possess the important and essential 
element of proportion and freedom from exaggera- 
tion, but exactitude is never absent from a drawing 
disclosing understanding, and conscientious treat- 
ment. 

There may seem little or no analogy between 
landscape and microscopical painting, but the same 
principles are involved points of sight, effective 
light, general entourage possibly a " preparation," 
dealing with unusual and unexpected complications 
of line, embracing physiological difficulties, requir- 
ing delicate conditions of luminosity, demands a 
deeper judgment, for it is often necessary to pre- 
pare the mind by careful and prolonged study, 
before the paper is touched ; especially in consider- 
ing and anticipating difficulties of representation, 
and how they may be overcome delicate structures, 
under the most careful illumination, often appear as 
streaks of LIGHT, when a slight touch of the con- 
denser may reveal distinct lines. These are points 
to be studied ; in fact, the subject should be " gone 
over " and arranged in all particulars, so that it may 
not outstrip the power of the pencil. All materials 



ON MICROSCOPICAL DRAWING AND PAINTING. 177 

should be of the finest quality, the paper, hard, thin, 
smooth, and unglazed ; delicate pencil drawings 
may be made on Bristol board, but such or any 
glazed or hot-pressed surfaces are totally unfitted 
to take colour. Fine drawing paper is preferable, 
when blocks are used each surface must be ex- 
amined for imperfections with a hand lens ; a 
delicate painting may be ruined, at a critical point, 
by an imbedded hair, an abrasion, or minute speck ; 
in the manufacture of these blocks, it has been 
found that in cutting up and folding the paper the 
true surface is not in every piece placed uppermost. 
For important work it is safer to select sheets 
strained in the usual way, in a small-sized folding 
drawing frame. Paper improves by age. If of 
undoubted antiquity, it fetches high prices. It is 
impossible to render satisfactorily, on a white sur- 
face opaque preparations showing minute injected 
anastomosing veins, arteries, or glands, the dark 
interstices separating them, cannot be drawn, or 
picked out, without sacrificing the regularity or 
destroying the uniform diameter of the vessels 
but such subjects may be effectively painted on a 
dull black paper, which may be previously pasted 
on a drawing block under pressure, using opaque 
or body colour, vermillion, yellow ochre, Antwerp 
blue, and carmine, combined with and regulated 
for substance and tint, with zinc white and gum 
water. Payne's grey, with zinc white, produces 
the peculiar shadowy hyaline tone so often seen as 
a substratum in such preparations where semi- 
transparent spongy tissues are involved fine effects 
of receding distances in following the depths of 
structures, may be produced by its use. Numerous 
12 



178 METHODS OP MICROSCOPICAL EESEAECH. 

brushes of sable are required, the hairs short and 
coming to a fine point, they should be of the best 
make, no brush that has touched Indian ink can be 
used for delicate colour, and those employed for 
carmine, yellow, and blue, should be marked and 
kept distinct ; the same applies to pens, often re- 
quired, but the pen carrying colours must be used 
with extreme discretion. If a fine line can be 
obtained with the sable, it is of higher quality, 
moreover with the handy pen the temptation is 
great to obtain hurried results by strokes and dots, 
but for pure black and white memoranda, or repre- 
sentations requiring speed, nothing can equal a fine 
pen charged with indian ink or neutral tint, re- 
membering never to approximate or cross a line 
until it be properly dry ; with this precaution a pen 
drawing may approach the semblance of an etching. 
The colours should be dry cakes, the palette pre- 
served as pure as possible ; moist pigments rubbed 
from pans become contaminated, and even dry 
cakes should be kept separated ; loose and in con- 
tact they chip and soil each other. Quality is all 
important, use only those which are " transparent." 
Manuals on painting contain lists of recommended 
pigments and their qualities, and generally receive 
no attention ; for the work in question it may 
remove difficulties to remember that important 
colours are neutral tint, Payne's grey, Antwerp 
blue, carmine, scarlet lake, yellow ochre, Hooker's 
greens, Nos. 1 and 2, and raw Sienna colours to 
avoid, vermillion, cadmium, the umbers, emerald 
green, and Vandyke brown. These are densely 
opaque, and " load " too heavily for delicate work ; 
a good test is to rub a portion of each cake of a 



ON MICROSCOPICAL DRAWING AND PAINTING. 179 

well-furnished box on a clean porcelain palette, side 
by side. When dry, those which appear dull and 
dusty (however useful they may be in landscape in 
large thin washes) reject. Everything may be 
accomplished with the remainder. There is no 
difficulty in conducting a painting by artificial light 
when conversant with the character and combina- 
tions of the few colours really required, a precau- 
tion, however, is necessary in painting tissues 
stained artificially with logwood or aniline dyes. 
These colours are very deceptive, and differ in 
appearance under degrees and qualities of light. 
Logwood stain (often used) in daylight has a blue 
tinge, under the lamp it appears as a decided port 
wine tint, and a difficulty may (in fact, does) ensue 
in matching day and lamplight work. When 
the entire subject has to be painted in the same 
tone, cakes of " mauve " and " aniline blue " now to 
be procured may be used alone, and thus stained 
tissues can be painted under any conditions of light 
without falling into error. It need hardly be said 
that such abnormal colours are to be used exclu- 
sively for those special preparations, and should 
never enter into the composition of, or even touch a 
general palette required for natural representations. 
Indian ink must be of superlative quality, the 
difference in price, although not deadly, is great. 
A piece should be secured, regardless of cost, and 
treasured. 

With practice in cultivating accuracy of touch, 
certainty of line, and ignoring the existence of 
{i rubber" and knife-edge, no difficulty need be 
anticipated in drawing on wood, zinc, or litho- 
graphic stone. 



ISO 



CHAPTER VIII. 



On Photo = Micrography. 

IT is not intended to claim any originality for the 
subject of this chapter, since the application of 
photography to the delineation of microscopical 
preparations is almost as old as the photographic 
art itself, extending back even to the days of the 
daguerrotype. Microscopists of the present gene- 
ration should think of this, and while paying tribute 
to the patient perseverance with which their fore- 
runners must have worked under all sorts of dis- 
advantages, should blush that, notwithstanding all 
the recent advances and all the simplicity of the 
gelatino-bromide process, so few avail themselves 
of the facilities it affords for the truthful and beau- 
tiful delineation of the objects of their study. The 
chief reason for this neglect is probably the idea 
among the uninitiated that photography is a very 
complicated and difficult art, dependent upon a very 
uncertain condition in our climate bright day- 
light and that unless one had the necessary 
day-time leisure and were expert in ordinary 
photography it would be useless to attempt this 
special application of the art. It is to expose the 



ON PHOTO-MICROGRAPHY. 181 

fallacy of all this, and to show that any micro scopist 
armed with a small text book and with a simple 
apparatus which it is quite within the bounds of 
possibility for him to make for himself, and having 
no leisure time but the dark winter evenings, can, 
after a few weeks' practice, produce pictures of 
specimens in his collection which, for absolute 
fidelity and beauty, are incomparably superior to 
the highest flights of the draughtsman's skill. In 
taking up the study of photography a beginning 
must be made somewhere, and the tyro's first efforts 
may as usefully, and with as great a prospect of 
ultimate success, be directed to this branch as to 
any other. It is a matter, not of doubt, but of cer- 
tainty, that his first attempts will be failures, from 
under exposure, over exposure, forgetting to draw 
the slide and therefore no exposure fog, frills, 
stains, pinholes, under - development, and other 
causes ; but had he commenced with portraiture or 
landscapes, he would have had the same dismal 
record of good plates gone wrong, and would have 
had the additional gratification of many an unpro- 
ductive tramp. Therefore we would say be not 
deterred from taking up this work because you are 
not a photographer. Commence operations and 
become one. It will, of course, be impossible here 
to give any elementary instruction in photography 
pure and simple. All that can be done is to describe 
such apparatus and processes as are special to this 
particular work, for all that is general a good 
text book of photography must be consulted. 1 

1 Captain Abney's "Instruction in Photography," Piper and 
Carter, is one of the best, but there are several well known 
and more modern text books from which choice can be made. 



182 



METHODS OF MICROSCOPICAL RESEARCH. 



The apparatus employed is simple. It consists of 
a microscope of any ordinary construction, a power- 
ful source of light, and a camera. The complete 
apparatus perfected and manufactured by Messrs. 
Swift & Son, Tottenham Court Road, from whom 
price, description, and all particulars may be 
obtained, is here illustrated. The mirror of the 




microscope is discarded except for special purposes, 
because the loss by reflection is very serious. The 
microscope is placed horizontally in a line with the 
source of light, and with its tube inserted by a 
light-tight joint into the front of the camera, which 
is supported at such a height that its centre 
coincides with the optic axis of the microscope. 
The object is held on the vertical stage by means of 
spring clips, and the light from the lamp is con- 
densed on it by one or more condensing lenses. 
There are two chief methods in use. In one the 
eyepiece of the microscope is removed, and the 
inverted image is received on the sensitive surface 
where it is first formed. In the other the eyepiece 
is retained in its place, and the image first formed 
in its interior is formed again with additional 
magnification by the aid of the eye-lens. By the 



ON PHOTO-MICROGRAPHY. 183 

use of the eyepiece the advantage is gained that any 
ordinary camera may be used, and in consequence 
of the shortness of the distance between the 
focussing screen and the microscope, the various 
adjustments of the latter are accessible, while 
focussing, without any special arrangements. In 
order to secure the same amount of magnification 
or to cover the same sized plate, when the eye- 
piece is not used, the camera must be of special 
construction so as to be capable of extension to two 
or three times the former distance, and when so 
extended to say a yard the focussing screen can 
only be reached by rods and bands or intermediate 
gearing. For facility, therefore, the eyepiece 
method is commendable, but the impairment of 
the image and the loss of light due to the inter- 
position of two additional un corrected lenses are 
so considerable that we would advise the removal 
of the eyepiece for all but special purposes, such 
as the covering of a very large plate. 

The microscope may be of any ordinary construc- 
tion that will allow the body to be placed hori- 
zontally, and it should have a stop to prevent it 
going beyond that position. It should be provided 
with a coarse adjustment, by rack and pinion, and 
a very sensitive fine adjustment for slow focussing. 
Very much of the success of the work depends upon 
the excellence of the slow motion. It is always a 
very important part of the microscope, but for 
photo-micrography it becomes especially so. It 
should be free from the slightest lateral motion, 
lest, in focussing, the image be removed from the 
centre of the plate, a very small displacement of 
the object glass being sufficient to effect this, when 



184 METHODS OF MICROSCOPICAL RESEARCH. 

working with liigli powers and a long camera. It 
must work with perfect smoothness and without 
loss of time in either direction. If the tube of the 
microscope can be shortened by unscrewing the 
part above the fine adjustment so much the better, 
for when working without the eye-piece, a long 
tube, especially if it be a narrow one, greatly 
contracts the field. This is one objection to the 
Jackson-Lister form of stand for photographic 
work, and the difficulty here can only be got over 
by selecting an instrument with a wide tube. There 
is another objection to the retention of a long tube, 
whether the eye-piece be employed or not. It is 
certain to give rise to a " flare " of light by reflec- 
tion from its inner surface, and flare, whether 
arising from this source or from the setting of the 
object glass, or from the interior of the camera 
itself, is absolutely fatal to the production of clean 
pictures, and results in the diffusion of a uniform 
light all over the plate, which impairs the purity of 
the shadows and produces a general fog. One chief 
seat of this internal inflexion is the fine adjustment 
tube. No amount of dead blackening, or even 
lining with black velvet will completely stop it. 
The only thing to be done is to interpose along the 
course of the tubes of the microscope, and in the 
camera, and even in the object glass itself, if neces- 
sary, a series of diaphragms. These may be cut 
out of cards with gun punches, and glued to narrow 
rings of cork to give them a grip of the tube. They 
and the corks also must be painted dead black with 
water colour (lamp black), and their number, 
position and aperture so adjusted that a line drawn 
from the centre of the object glass to the edge of 



ON PHOTO-MICROGRAPHY. 185 

the tube when at its shortest, shall just touch the 
edges of all these apertures. Thus arranged they 
will not contract the field, and will not allow a ray 
of light to fall on anything but the front faces of 
the diaphragms themselves, whence it cannot be 
reflected to the plate. \Vhen the eye-piece is used 
the diaphragms must be differently adjusted, for 
the tube then practically ends at the front surface 
of the field glass, and its diameter is practically 
the clear aperture of that lens. A slight sliding 
backward or forward of the diaphragms will be 
sufficient to effect this adjustment. In the camera 
itself the diaphragms, if required, will take the 
form of sheets of blackened card with central aper- 
tures. The writer, who works with quarter plates 
(4J- by 3|- inches), prefers to make the aperture 
nearest the microscope small and circular, the next 
larger and shortly oblong, with very rounded 
corners, the next more oblong and with less rounded 
corners, and the last oblong with acute corners and 
the shape of the plate, but a trifle smaller. On 
looking through the whole length of over a yard of 
camera and tubes when so arranged, and with a 
blaze of light streaming in from the condensers, 
not a single stray beam can be seen, and nothing is 
visible but the object glass " full of light." Then 
when a plate is properly exposed and developed, 
the deepest shadows come out as clear as the glass 
itself, and the negative prints brightly and quickly. 
When working with 3, 2, or even 1 inch objectives, 
the fine adjustment may be dispensed with, and 
then, if the microscope be of the old Ross pattern, 
the tube may be entirely discarded, and the objec- 
tive screwed (by means of a short adapter, if need 



186 METHODS OF MICROSCOPICAL RESEARCH. 

be, or simply wedged) into the arm that usually 
carries the tube. By this means the field is only 
limited by the aperture of the objective, and there 
is no possibility of flare from the tubes. 

A mechanical stage, with concentric rotation, 
will greatly facilitate the adjustment of the picture 
in the centre of the plate. With objectives of 
higher power than -| inch, these mechanical motions 
become indispensable. Spring clips are required 
to keep the object in a vertical position. 

The camera may be of a size to take plates from J 
to whole plate size according to the worker's choice, 
but at first, at all events, if not permanently, the 
smaller sizes are best. The quarter plate size is 
most generally used, and plates of that size are 
always obtainable and are cheap, but we would 
recommend 5 by 5 inches as a more useful size, con- 
forming more nearly to the circular form of the 
field, and to many objects, diatoms, echinus spines, 
sections of stems, &c., which involve a great waste 
of surface when taken on oblong plates. By means 
of a simple adapter a camera of this size will take 
^ plates when desired, and these can be placed with 
their longer edges either horizontal or vertical. 

The camera should be made with a bellows body 
capable of closing up to 6 or 7 inches and extending 
to about a yard, it will then be available for use, 
either with or without the eye-piece. To the front 
should be fitted the base of a smaller bellows of 
conical form, whose small end terminates in a wood, 
metal or cardboard ring, lined with black velvet 
and fitting smoothly and light-tight on to the 
outside of the tube of the microscope. The dark 
slide which carries the plate should be single, and 



ON PHOTO-MICEOGEAPHY. 187 

constructed to take wet plates as well as dry. 
Unless a very large camera or a very small micro- 
scope is used, the camera will probably want block- 
ing up to bring its centre exactly to the same height 
as the optic axis of the microscope. For this pur- 
pose a light support of pine should be constructed. 
It should be as long as the camera when fully 
extended, and should be graduated in inches along 
one edge. If polished it will keep cleaner and look 
much better than if left plain, and will not wear 
the edges of the bellows. On the top of this 
support the two ends of the camera may be made 
to slide, and may be secured in any position by set 
screws running in a central groove. 

The microscope, camera, illuminating apparatus, 
&c., are to be clamped down in marked positions 
on a base board, about 6 feet long, 11 inches wide, 
and 1 inch thick. The point of the base board, 
immediately below the position of the object when 
in use, should be marked, and the board should be 
graduated in inches from this point in both direc- 
tions. The camera support should have a play of 
five or six inches to, or from the microscope, and 
when adjusted, should be clamped by a single turn 
of each of two screws, which hold it to the base 
board. To meet the wants of those who are 
content to use the eye-piece, Mr. Stanley, of London 
Bridge, has constructed a cheap apparatus, con- 
sisting of an ordinary J plate bellows-camera, and 
a base board, to carry microscope and lamp, and 
with adjustable platform to bring the camera up to 
the height of any ordinary microscope. 1 

1 Mr. Stanley also publishes a pamphlet, containing instruc- 
tions for working dry plates, silver printing, &c., which is 
worthy the attention of beginners. 



188 METHODS OF MICROSCOPICAL EESEAECH. 

As to illumination, there is a great choice, but we 
may at once dismiss for ordinary work : Sunlight, 
as being too precarious, and necessitating mid-day 
leisure ; Magnesium, as too expensive, and very 
difficult to focus by, on account of its trick of going 
out when left to itself. Electric arc necessitates the 
charging and discharging of 30 or 40 Grove's cells, 
a pleasure that can be appreciated only by those 
who have tried it. In favour of Incandescent lamps 
we cannot say a word. A 20-candle Swan lamp 
requires as powerful a battery as a small arc, and 
has none of its advantages. Its light is not con- 
centrated, it is feeble and yellow. Lime light is the 
cheapest, least troublesome, and, on the whole, the 
best of powerful artificial illuminants. Our choice 
is therefore limited to lime-light, gas and paraffin. 
Of these, the latter is everywhere obtainable, gives 
a white light, and is all that could be desired for 
ordinary work with objectives up to -|- inch. Our 
own arrangement consists of a paraffin lamp with 1^ 
inch wick placed close to one end of an oblong zinc 
reservoir. It is supported on a block about 20 
inches from the stage. The glass chimney is narrow 
to allow of the close approximation of a plano-convex 
condensing lens of 3 inches aperture and 3 inches 
focus, which collects the light and transmits it in a 
slightly convergent beam. At a distance of 5 inches 
from the stage is placed a second plano-convex 
lens of 3 inches aperture and 5 inches focus. This 
further converges the beam on the object, and gives 
a brightly and uniformly illuminated disc of about 
f of an inch in diameter, so that large objects can 
be well photographed under low powers. The 
object of using a short focus lens near the lamp is, 



ON PHOTO-MICROGRAPHY. 189 

of course, to intercept as large a quantity of light 
as possible, while the longer focus lens nearer the 
stage secures a larger disc than would be possible 
with a lens of shorter focus. The convex sides of 
both the lenses are turned towards each other. 
When working with the \ inch and higher powers, 
a further concentration of the light is effected by an 
achromatic condenser. If the edge of the flame is 
turned towards the condensers, the light is very 
bright, but there is a difficulty in illuminating a 
large field uniformly. If, however, the flame make 
an angle of about 5 degrees with the optic axis, this 
difficulty disappears without sensibly reducing the 
brightness of the field. 

The arrangement in which an optical lantern 
replaces the simple lamp may be used with the 
advantageous results of shortening exposures, and 
facilitating focussing under high powers. 

When working at night, there is no necessity to 
use a focussing cloth. The image is first received 
on a screen of the finest possible ground-glass, made 
more transparent by waxing the ground surface, but 
even this is too coarse for delicate focussing, and to 
get the final focus the screen is removed and 
replaced by a lens of about 1 inch focus held in the 
hand, but attached to a strip of wood, whose arms 
are brought up against the end of the camera when 
in use, so that, in shifting it about to examine 
different parts of the image, it is constrained to 
remain in one plane, and its distance has been so 
adjusted once and for all, by previously focussing 
the ground-glass when in position, that its focal 
plane is exactly that occupied by the sensitive plate. 
Mr. Stanley's arrangement for focussing consists of 



190 METHODS OF MICROSCOPICAL RESEARCH. 

an ordinary ground-glass screen, with cover glasses 
cemented on to the ground side here and there, so 
that at these places the screen becomes almost as 
clear as plain glass, and parts of the image can be 
examined through them with an ordinary focussing 
lens. When the apparatus is used without the 
eye-piece, focussing is effected by turning a rod 
running underneath the camera, and actuating a 
roller round which, and also round a groove in 
the fine adjustment screw above, an elastic band 
passes. 

The possible non-coincidence of the visual and 
actinic foci of the object glasses is a point that 
must be borne in mind, and each objective must be 
tested to ascertain to what extent, if at all, this 
non-coincidence occurs. This source of error is, 
however, usually eliminated by interposing, in the 
path of the beam between the two condensers when 
focussing, a glass cell with parallel sides, containing 
a solution of cuperic ammonic- sulphate 1 . This will 
cut off all the rays of low refrangibility, and in nine 
object glasses out of ten, will be found to supersede 
any other correction, at all events, when paraffin is 
the illuminant. "With the electric arc or other light 
in which the ultraviolet rays largely predominate, 
the case is different. The mode of testing for this 
want of coincidence, and ascertaining its amount, 
is to interpose the "copper" cell and obtain the 
best possible visual focus of some object with hard 
and sharp lines, with the glass to be tested. The 



i Prepared by adding ammonia to an aqueous solution of 
sulphate of copper, until the precipitate first formed is re- 
dissolved. 



ON PHOTO-MICROGRAPHY. 191 

cell is then removed, a photograph is taken, and if 
it shows any want of sharpness, the cell is again 
interposed, and by means of the fine adjustment 
screw, the object glass is approached a little nearer 
to the object, until the image as seen with the eye 
is about as indistinct as that on the photograph. 
Another plate is then exposed and developed, and 
will probably be quite sharp. If not, another slight 
alteration of focus must be made, and the process 
repeated if necessary, until at last the requisite 
sharpness is obtained. It should be then noticed 
how much the fine adjustment screw has to be 
turned, to bring back the proper visual focus, when 
the copper cell is used, and this amount should be 
recorded and applied as a constant correction when- 
ever the lens in question is used. 

So much for apparatus. Now we must say a few 
words about the photographic processes employed. 
Gelatine dry plates are the best suited to this work 
on account of their rapidity, the little apparatus 
required, the cleanliness of the process of develop- 
ment, and their being always ready for use. There 
are many excellent dry plates of various degrees of 
rapidity, and still more various prices in the market, 
and it is very much better to buy them than to 
make them for oneself. A rapid plate is most 
suitable, but there is no occasion for excessive 
rapidity. Gelatine plates must be exposed to none 
but deep red light, and the less of this they are 
exposed to the better. When unpacked they should 
be stored in grooved light-tight boxes. The smallest 
ray of white light finding access to the dark room 
will be fatal to success. If the operator works by 
night only, he will not need a special dark room. 
Any ordinary room with a closely fitting door, to 



192 METHODS OF MICROSCOPICAL EESEAECH. 

keep out lamp light or gas light from the adjoining 
apartment (if any), will answer, provided there is 
not a lamp within a few yards of the window, and 
the moon is not shining brightly. The most 
convenient light to work by is that afforded by a 
paraffin lamp, protected by a chimney of ruby glass, 
or a screen of good orange demy free from pin 
holes. This screen must be so arranged as not to 
allow a single ray of white light to enter the room 
in any direction, while it allows a proper supply of 
air to the lamp. If the operator wishes to work by 
daylight he must either darken a room or large 
cupboard entirely, and use the paraffin lamp as 
before, or he may glaze the window with ruby 
glass, and make it additionally secure by pasting a 
layer of yellow demy over it, or two thicknesses 
of bookbinders' red cloth may be used instead of 
glass and paper. In whatever way the dark room 
is lighted, the plate must be shaded from it as 
much as possible, and only brought into the full 
light for any length of time when development is 
nearly complete, and it is necessary to use all safe 
light to watch the completion of the process. The 
unpacking and storing of plates, and placing them 
in the dark slide, may be conducted in almost entire 
darkness. Before commencing work the following 
solutions should have been made up : l 



1 Since this article was originally written, photographic 
chemistry has made great advances; the above developing 
(&c.), solutions are here retained as good, but knowledge of 
and experience in the art will enable the operator to make 
choice of the modern formulae most suitable for his own 
.method of working. 



ON PHOTO-MICROGRAPHY. 



193 



For developing^ 



For hardening. 
For fixing. 



' L f Pyrogallic acid . . 

I Distilled water .. 

f Ammonia solution 
B ! Potassic Bromide 

I Distilled water . . 
p f Potassic Bromide 

( Distilled water . . 
-pv f Ammonia solution 

I Distilled water , 



Normal 
developer. 



For intensifying. 



36 grains ^ 
12 oz. 

I oz. 
30 grains 

6oz. 
15 grains 1 For over- 

1 oz. J exposure. 

1 drm. 1 For under- 



1 oz. J exposure. 
Alum saturated solution in common water, 
f Sodic hyposulphite, 4 oz. 
I Common water, 1 pint. 

Mercuric chloride saturated solution in 

common water. 
J Ammonia solution, 1 oz. 
1 Common water, 8 oz. 



(Hydrochloric acid, 8 drops. 
Forremovmgstams{ Common 



A beaker of about two and a half ounces capacity, 
a drachm measure graduated to minims (60th of a 
drachm-drops), a papier-mache tray, 4J by 3J, for 
developing, a porcelain dish to hold the hyposulphite 
of soda (" hypo "), another dish or tray of papier- 
mache or porcelain to hold the alum, a broad camel- 
hair brush, and an abundant supply of water should 
also be in readiness. Operations may then be com- 
menced by taking a plate from the box, and at once 
replacing the lid, passing the brush gently over its 
coated surface to remove particles of dust, and 
placing it in the dark slide, previously dusted inside, 
which is then to be shut up. Neither in this nor in 
any other operation must the sensitive surface be 
touched with the fingers. The slide is then carried 
to the camera, placed in position, and the exposure 
given. This, however, must not be done by simply 
drawing the shutter, for in this act vibrations are 
13 



194 METHODS OF MICROSCOPICAL RESEARCH. 

inevitably set up in the apparatus, and the plate, if 
exposed during this vibration, would not be impressed 
with a sharp image. Before drawing the shutter a 
slip of card, covered on both sides with velvet, and 
attached to a separate stand, should have been 
interposed in the beam of light between object and 
object glass, if the latter is of 1^ inches or lower 
power; behind the stage if of higher power. With 
this stop in position the shutter may be drawn 
without exposing the plate. A few seconds are 
then allowed for vibrations to subside ; the stop is 
taken in the hand and held for a second touching 
nothing, and then quickly removed and the exposure 
commenced, the time being noted. If the exposure 
is only to be of a few seconds' duration, the operator 
still retains the stop in his hand and remains per- 
fectly still while he counts the seconds on a watch, 
or the vibrations of a ball attached to a string 39 
inches long, and held in his other hand. The 
instant the time expires the stop is replaced, the 
shutter closed, and the slide withdrawn and carried 
to the dark room; 2oz. of A solution are then poured 
into the beaker (there is no need to be particular to 
half a drachm or so, and it is easy to guess when 
sufficient has been poured into the beaker). The 
light is then turned very low, and the operator, 
turning his back to the lamp, removes the plate, 
passes the brush over it once more, and places it 
face upwards in the developing dish ; then the 
"pyro" solution from the beaker is poured on to the 
plate with a steady and rapid motion, so that the 
whole plate is flooded at once. The tray is then 
put aside in a shady place for a few minutes, while 
the operator measures out 25 drops (minims) of B. 



ON PHOTO- MICROGRAPHY. 195 

Here again an excess or deficit of three or four 
drops is of no consequence. This solution is then 
to be poured into the beaker, into which also the 
" pyro " solution from the tray is to be poured, and 
the mixture is then returned to the tray in an even 
wave, and kept gently rocking. For a few seconds 
nothing will be visible, and for that time it is quite 
as well to shade the plate from the light, then, if 
the exposure has been correct, gradually the highest 
lights will appear and get deeper and deeper, and 
detail will begin to appear in the lightest half 
tones. If all is going well, the plate may be left 
at this stage while an additional 50 drops of B 
are measured out and poured into the beaker. 
Again the solution from the tray is to be returned 
to the beaker, and again the mixture poured back 
and the tray kept rocking. 

By this time the picture should be full of detail, 
and more light will be required, and may be safely 
used. This is a most important time, and great 
judgment is required to know when to stop the 
development. In the feeble light employed, it will 
seem as if the picture is blackening all over and 
disappearing, but really, if properly exposed, it is 
only gaining density. The judgment will be assisted 
by removing the plate from the tray, and holding it 
up before the light ; the amount of density can then 
be better seen. If judged insufficient, the plate is 
returned to the tray and rocked a few seconds 
longer. If the exposure has been properly timed, 
the whole development will occupy about two and a 
half or three minutes from the first application of 
the B solution. When at last the proper moment 
arrives, the plate is to be removed from the tray, 



196 METHODS OF MICROSCOPICAL RESEARCH. 

and well swilled with water from a jug or tap. It 
is then to be placed face upwards in the dish of 
" hypo," and left there five minutes or so, until all 
trace of milkiness disappears from the back of the 
plate. It may then be removed from the "hypo," 
again swilled with water, and if the weather is not 
hot, and the plates are known to have no tendency 
to frill, it may be placed in a dish of water and left 
to soak. This soaking must be continued for about 
six hours, in several changes of water. The plate 
may then be stood on end to dry. Heat must not 
be applied to hasten the drying, or it will cause the 
gelatine to dissolve. From the time the plate comes 
from the hypo it is insensitive to light,, and, indeed, 
it may be taken into the light and fixed there as 
soon as the developer is washed off, without any 
considerable harm happening to it, but it is best not 
to expose it to light until it is fixed. The photo- 
grapher cannot be sure of the character of his 
negative until it is fixed, and during this process it 
will undergo a remarkable change, it will lose its 
translucency and become transparent. If the 
exposure and development have been correct, the 
highest lights will be so dark as to make it very 
difficult to distinguish even bright objects through 
them, while the deepest shadows should be almost 
as clear as the plain glass, and the half tones full of 
detail. 

If on the first application of B, the details come 
up with undue rapidity and then begin to fade 
away, the plate has been over-exposed, and the 
developer must be instantly poured back into the 
beaker and the plate flooded with water while 20 to 
30 drops of C, according to the amount of over- 



ON PHOTO-MICROGRAPHY. 197 

exposure, are measured out and added to the 
contents of the beaker, which are then to be 
returned to the plate after the water has been 
poured off. This will retard the development and 
preserve the purity of the shadows, while the high 
lights gain in density. After a time 30 or 40 drops 
of B may be added to complete the development. 
Under-exposure is indicated by slowness in the 
appearance of detail in the shadows and half tones 
after the full quantity of B has been added, and is 
remedied by the addition of 10 to 20 drops of D to 
the developer. In all cases when additions have to 
be made to the developer, the additional substance 
should be poured into the beaker, and the solution 
from the plate added to it. This will secure a 
uniform mixture before the developer is returned to 
the plate. If the additions were simply poured 
directly into the tray they would act locally and 
spoil the picture. The operator must learn to 
recognise an under- or an over-exposed plate by its 
character after development as well as before. 
Over-exposure results in thin (i.e., weak or trans- 
parent), flat, soft negatives, full of detail, but 
deficient in intensity, while an under-exposed and 
forced plate yields a dense and harsh negative with 
little detail. It is useful to know this, because one 
may occasionally wish to give the negative a soft or 
a harsh character to suit some special subject. The 
rule is, for violent contrasts, under-expose and 
force in development ; for a finely-graduated image 
over-expose, and use the bromide freely. For most 
purposes we should avoid either extreme. 

If the films have a tendency to frill (i.e., to 
pucker and leave the glass at the edges) or blister 



198 METHODS OF MICROSCOPICAL RESEARCH. 

during or after development and in very hot 
weather all plates have these tendencies they must 
be soaked for about five minutes in the alum solu- 
tion, both before and after fixing, to harden the 
films. In hot weather one should not wait for the 
commencement of frilling or blistering before apply- 
ing the remedy, but take it for granted that it will 
occur, and pass every plate through the alum. It 
can do no harm in any case. 

Plates developed with " pyro " acquire a pale 
brown or yellow colour, which, if strongly marked, 
it may be advisable to remove by a few minutes' 
immersion in the hydrochloric acid solution. Over- 
exposed or under-developed plates can be intensified 
as follows : The mercuric chloride solution is first 
applied and kept in motion until the negative 
becomes a very pale grey, almost white. If the 
operation be conducted in a black dish the negative 
will appear as a most beautiful positive when the 
whitening is complete. The plate should then be 
thoroughly washed for at least half-an-hour in 
several changes of water. The weak ammonia 
solution should then be applied, and in a few 
seconds will blacken the image. It will then want 
a thorough washing in several changes of water, 
prolonged through about six hours, after which it 
may be stood in a rack to dry. 

When dry, the negatives must be varnished with 
photographic varnish flowed over them while hot. 
This will protect the films from mechanical injury 
aud from silver stains in printing. When negatives 
have been intensified, it is especially necessary to 
varnish them as soon as possible, as they are liable 
to turn white by exposure to the air. 



ON PHOTO-MICROGRAPHY. 199 

It is very difficult to lay down any general rule 
for length of exposure, since there are so many 
variables thickness and colour of the object, 
colour of the medium (balsam especially sometimes 
acquires a yellow colour highly objectionable), focal 
length and angular aperture of the object glasses, 
and the number of lenses and kind of glass of which 
they are composed, length of camera, rapidity of 
plates, presence or absence of eye-piece, and mode 
of illumination, all have to be taken into account to 
give some idea of what may be expected ; we append 
particulars of a few of our negatives. The first 
three were taken with an A eye-piece, and a lamp 
having a 1 inch wick only. The plate was about 12 
inches from the object. 

" Tongue " of Blowfly. Balsam. Two inch objec- 
tive by Stanley. Home made plate. Five min. 
Ferrous oxalate developer. Under-exposed. 

Diatom. Coscinodiscus Excavata. ^ inch objec- 
tive and achromatic condenser. Home made plate. 
10 min. Pyro. Correct exposure. 

Diatom. Auliscus sculptus. % inch objective by 
Beck, and achromatic condenser. Edwards' plate. 
26 min. Pyro. Rather over-exposed. 

The following were taken without an eye-piece, 
and with a lamp having a 1^ inch wick. The 
distance of the plate from the object is given in 
each case. 

Tongue of Gat injected carmine and stained. Trans- 
verse Section. Balsam. 3 inch objective of 12 by 
Browning. Distance 12f inches. Miall's plate. 10 
sees. Pyro. Correct exposure. 

Rachis of Pteris Aquilina. Transverse Section un- 
stained. Gly. jelly. 2 inch objective by Stanley. 



200 METHODS OP MICROSCOPICAL RESEARCH. 

Distance 22-J- inches. Miall's plate. 1 min. Pyro. 
Over-exposed. 

Human Kidney injected carmine. Transverse 
Section in Balsam. Object yellowish. 1 inch objec- 
tive by Stanley. Distance 36 inches. " German ' 
plate 1^ min. Oxalate. Correct exposure. 

" Tongue " of Blowfly. Balsam. J inch objective 
and achr. condenser. Distance 40 inches. Lancaster's 
plate. 1^ min. Oxalate. Correct exposure. 

Spicules of Synapta. Dark ground illumination 
by spot lens. 1 inch objective by Stanley. Dis- 
tance 41 inches. " German " plate. 2 min. Oxalate. 
Correct exposure. 

Salicine. Polarized light. Crossed nicols. With- 
out selenite. 1 inch objective. Distance 20 inches. 
Home made plate. 2^ inin. Oxalate. Correct 
exposure. 

Ctenoid scales. Skin of sole. Dry, as opaque 
object. White, but under cover-glass, and therefore 
loss of light by reflection. Light condensed as 
usual, but incident at about 10. 3 inch objective of 
12 by Browning. Distance 28 inches. Miall's plate. 
5 min. Pyro. Correct exposure. 

These examples have been selected as illustrative 
of very various objects, magnifying powers and 
modes of illumination. Opaque objects are the 
most difficult, on account of the trouble of focussing 
by the small amount of light they reflect. 

Although any object glass may be used for this 
work, some possess qualities which specially fit 
them for it, and there are others in the same degree 
unsuitable. The most important properties of an 
objective for photographic work good definition 
being, of course, pre- supposed as essential for all 



ON PHOTO-MICROGRAPHY. 201 

purposes are penetration and flatness of field. 
These qualities are of greater importance in lenses 
for photographic than for visual purposes, for in 
viewing an object under the microscope the observer 
has the power of focussing in rapid succession, and 
by imperceptible gradations, points at different 
depths and different distances from the centre of 
the field; but a photograph represents only such 
structures as were in focus at the time of exposure, 
and once taken, the focus is unalterable. It is 
therefore desirable to secure as great a depth of 
focus and as flat a field as possible qualities which 
are incompatible with large apertures. 

This brief chapter is merely elementary and 
intended as a guide to beginners. Those who desire 
to thoroughly acquire and successfully practise this 
fascinating art, and to learn the details of the most 
recent processes and manipulations, as well as the 
use and value of apochromatic objectives and of 
all other modern appliances and apparatus, should 
obtain " PRACTICAL PHOTO-MICROGRAPHY" recently 
issued by that master of the art, Mr. Andrew 
Pringle, and published by Messrs. Iliffe and Son, 
St. Bride Street, London, E.G. 



THE END. 



14 



202 METHODS OF MICROSCOPICAL RESEARCH. 



In bidding farewell to his readers the author 
ventures to express the hope that the results of his 
thirty years' experience in microscopy and micro- 
scopical manipulation, as embodied in this treatise, 
may tend to simplify the work which has lost none 
of its interest or attractions for him, and that those 
who may be induced to prosecute this delightful 
pursuit may be enabled thereby to far surpass any 
results or achievements upon which he may venture 
to consider he is entitled to congratulate himself. 
At least he may affirm that his work has been 
always prosecuted with the most sincere desire and 
endeavours to produce the best results and to 
advance true microscopical science. 



INDEX. 



ABSOLUTE alcohol, hardening 

tissues by, 49. 
Acetate of potash, 103. 
Adenoid tissue, 58. 
Alcohol, as a hardening agent, 41 ; 

dilute, 54. 
Algae, fluid for mounting, 104 ; to 

clean diatoms on, 146. 
Alum cochineal staining, 86. 
Ammonia carmine staining fluid, 

82. 

Ammonium bichromate, 46. 
AmMin blue black, 86. 
Anilin colors as stains, 87 ; list of, 

88. 

Anilin stainings, clearing, 91. 
Animal tissues, preparation of, 38. 
Animal, to inject an, 71, 73. 
Apparatus,list of , for laboratory, 32. 
Aqueous humour of eye, 16. 
Aqueous injection fluids, 65. 
Art, microscopical, 7. 
Asphalt varnish, 107. 
Astigmatism, 21. 



B. 



BACILLI and bacteria, staining, 
91, 92, 93, 94; Rev. Gordon 
Thompson's process, 94. 

Balsam, Canada and xylol, 78. 

Beale, Dr., on instruments and 
their uses, 27; on methods of 
preparation, 6. 



Beetle, muscle of, 49. 

Bell's cement, 106. 

Bichromate of potass ; 45. 

Binocular vision, 24, 25. 

Bismarck brown, 89. 

Blood vessels, 57 ; injection of, 61, 

69. 
Blue injection mass, 64 ; fluids, 

66, 67, 68. 
Borax-carmine staining fluid, 83. 



C. 



CAMERA lucida, in microscopical 
drawing, 167. 

Canada balsam, 99 ; mounting in, 
133 ; diatoms in, 149. 

Capillary vessels, 61 ; develop- 
ment of, 59 ; injecting, 69. 

Carmine injection fluids, 65, 66; 
mass, 62 ; staining, 78 ; fluids, 
Beale's, 82; Huxley's, 83; 
Eanvier's, 82. 

Carrot, embedding in, 111. 

Cartilage cells, 56. 

Cathcart microtome, 127. 

Celloidin embedding, 117. 

Cement, BeU's, 106; dammar, 
105; gelatine, 106; gold size, 
107 ; gutta-percha, 107 ; marine 
glue, 107 ; mastic, 109 ; shellac, 
107 ; white zinc, 107. 

Cement substance of muscle, 58. 

Cements, 105. 

Chemicals for laboratory, list of, 
34. 



204 



INDEX. 



Chloride of gold, 59. 

Choroid coat of eye, 16-18. 

Chromate of ammonium, 46. 

Chromic acid, 42 ; and alcohol, 
43 ; and nitric acid, 55. 

Chrysoidin, 93. 

Ciliated epithelium, 54. 

Clearing media, 95 ; (on) and mani- 
pulation of sections, 96. 

Clearing with xylol and phenol, 
95 ; anilin stainings, 91 ; clove 
oil, 97 ; bergamot oil, 98 ; cedar 
wood oil, 98 ; turpentine, 97. 

Cochineal staining fluid, 86 ; 
alum, 86. 

Colour, 23. 

Colour-blindness, 23. 

Colours for use in microscopical 
drawing, 177, 179. 

Conversion of French fluid mea- 
sures into English ditto, 36; 
weights, 37. 

Cornea of eye, 15, 57, 59. 

Crystalline lens of eye, 1, 16, 17. 



D. 

DAMMAR varnish, 105. 

Deane's (mounting) medium, 101. 

Developing fluids for photo-micro- 
graphy, 192. 

Developing photo-micrographs, 
194. 

Development of capillaries, 59. 

Diatomacese, the preparation of, 
144; to clean, 146, 147; to 
mount in balsam, 149 ; to 
mount " dry," 148 ; mounting 
stage for, 152 ; to mount in 
styrax, 157. 

Double staining vegetable sections, 
142 ; with hsematoxylin and 
eosin, 89; with picro-carmine, 
144. 

Drawing and painting (micro- 
scopical), 160. 

Dry" slides, 148. 



E. 



EHELICH'S hsematoxylin staining 
fluid, 79; staining for bacilli, 93. 

Embedding, 109 ; in carrot, 111 ; 
celloidin, 117 ; gum and gly- 
cerine, 116 ; paraffin, 113 ; pith, 
112 ; wax and oil, 113. 

Endothelium, 56, 57. 

Eosin, 89. 

Epithelium, 5. 

Examples in photo-micrography, 
199. 

Eye, the human, essay by Geo. 
E. Davis, F.R.M.S.,F.L.S., &c., 
13 ; aqueous humour of, 16 ; 
choroid coat of, 16, 18 ; cornea 
of, 15 ; crystalline lens of, 1, 16, 
17 ; imperfections in, 14 ; iris 
of, 15, 17 ; pigmentum nigrum 
of, 16 ; retina of, 16, 18 ; sclero- 
tic coat of, 15 ; vitreous humour 
of, 16. 

F. 

FAEEANT'S medium, 102 ; mount- 
ing in, 137. 

Fearnley, Dr., injecting apparatus, 
73, 74 ; freezing microtome, 126. 

Fill an ice microtome, to, 125. 

Fluid measures, French, conver- 
sion into English ditto, 36. 

Fossil deposits, to clean, 147; dia- 
tomaceae, 147. 

Freezing, 109. 

Frog, 56, 58, 60. 

Fuchsin and eosin, 87. 

G. 

GAGE'S picro-carmine staining 

fluid, 84. 
Gelatine, 61 ; cement, 106; and 

honey, 101. 
Gibbes', Dr. Heneage, stain for 

bacilli, 92, 
Glycerine, 53, 102; jelly, 100 ; 

(mounting in) 136 ;i Kaiser's, 

101; Lawrence's, 101; mounting 

in, 135. 



INDEX. 



205 



Goadby's fluid, 104. 
Gold chloride, 59. 
Gold size, 107. 
Green injection mass, 65. 
Gum and glycerine, 102 ; (embed- 
ding), 116. 

Gum and syrup, 118. 
Gum styrax, 105. 
Gutta-percha cement, 107. 



H. 

H^MATOXYLIN staining fluids, 79, 
80; Ehrlich's,79 ; Kleinenberg's, 
81; Mitchell's, 81; Weigert's, 
80. 

Haematoxylin and eosin, double 
staining, 89. 

Hardening agents, 38 ; tissues, 38 ; 
by absolute alcohol, 49 ; by 
alcohol, 41 ; bichromate of 
potass, 45 ; ammonia, 46 ; 
chromic acid, 42 ; and alcohol, 
43 ; chromate of ammonium, 
46; Miiller's fluid, 47; and 
spirit, 48 ; picric-acid, 50 ; osmic 
acid; 51. 

Higginson's syringe, 75, 76. 



ICE microtome, to fill an, 125. 

Illumination in microscopical 
drawing, 171; in photo-micro- 
graphy, 188. 

Injecting, methods of, 69; an 
animal, 71; by Dr. Fearnley's 
apparatus, 73, 74. 

Injection of blood vessels, 61, 69 ; 
with syringe, 69 ; capillaries, 69. 

Injection fluids, aqueous, 65 ; blue, 
66, 67, 68 ; carmine, 65, 66. 

Injection mass, 61 ; blue, 64 ; 
(Robin's), 64 ; carmine, Carter's, 
62; Stirling's, 62; Sims Wood- 
head's, 63 ; green, Scheeles', 65 ; 
red, 61. 

Injecting of various organs, 69, 70. 



Instruments and their uses, 1, 10, 

27 ; Dr. Beale, on, 27. 
Introduction, 1. 
Iodine green, 90. 
Iris of eye, 15, 17. 

K. 

KAISER'S glycerine jelly, 101. 
Kleinenberg's hsematoxylin stain, 
81. 

L. 

LABOBATORY, list of apparatus for, 
32 ; of chemicals for, 34. 

Lacteal, injection of, 49. 

Lawrence's glycerine jelly, 101. 

List of anilin colours for staining, 
88 ; apparatus required, 32 ; 
chemicals, 34. 

Logwood staining, 78. 

Lungs, 57. 

Lymphatic gland, 49 ; vessels, 61. 

Lymphatics, 58. 

M. 

MARGARINE crystals, 53. 

Mastic cement, 109. 

Membranous connective tissue, 
58. 

Methods of preparation, 5 ; Dr. 
Beale on, 6. 

Methyl green, 90 ; violet, 90. 

Micro-photography, 8. 

Microscope, the, 10 ; for work, 28 ; 
objectives for, 31; in micro- 
photography, 183. 

Microscopical art, 7. 

Microscopical drawing and paint- 
ing, 161; camera lucida in, 
167; colours for, 177, 179; 
illumination in, 171 ; paper for, 
177. 

Microtomes, 119. 

Microtome, Cathcart's, 127 ; 
Fearnley's, 126; Minot's, 129; 
Eutherford's, 121 ; Thoma, 129; 
Williams', 123. 

Minot's microtome, 129. 



206 



INDEX. 



Miscellaneous preparations, 53. 

Mitchell's logwood stain, 81. 

Monobromide of napthalin, 105. 

Mounting (on), 132 ; in Canada 
balsam, 133 ; Farrant's medium, 
136 ; glycerine, 135 ; glycerine 
jelly, 136; diatoms, 148, 149; 
insects, 138 ; polycystina and 
radiolaria, 157. 

Mounting media, 98 ; acetate of 
potass, 103 ; Canada balsam, 99 ; 
hardening of, 99 ; and benzol, 
99 ; and xylol, 99 ; Deane's fluid, 
101 ; glycerine, 102 ; jelly, 100 ; 
Goadby's fluid, 104 ; mono- 
bromide of naphthalin, 105 ; 
styrax, 105. 

Mounting medium for algae, 104. 

Mounting stage (Cole's), 152. 

Mouse, 60. 

Muller's fluid, 47 ; and spirit, 48. 

Muscle, 60. 

N. 

NAPHTHA and creasote, 104. 
Nerve ganglia, 60. 
Nitrate of silver, 55. 
Nitric acid, 42. 
Nuclei (of cells), staining, 77. 

0. 

OBJECTIVES for working micro- 
scope, 81. 

Oil of bergamot, 98. 

Oil of cloves, 97 ; cedar wood oil, 
98. 

Olfactory epithelium, 54. 

Omentum, 56. 

Orange and rubin, 91. 

Organ of vision in man, constitu- 
tion of, 1. 

Osmic acid, hardening by, 51 ; its 
uses, 51. 

P. 

PAPEE for microscopical drawing, 

177. 
Paraffin, embedding in, 113. 



Phenylamin, 93. 

Photo - micrographs, developing, 
194. 

Photo-micrography, on, 180 ; 
illumination in, 188 ; develop- 
ing fluids for, 193 ; examples in, 
199. 

Picric-acid, hardening by, 50; its 
uses, 56. 

Picro-carmine staining, 78 ; fluid } 
Gage's, 84; Weigert's, 84 ; double 
staining, 144 ; and eosin, 85. 

Picro-lithium carmine, 85. 

Pigmentum nigrum of eye, 16. 

Pith, embedding in, 

Polycystina, cleaning and mount- 
ing, 157. 

Preparation of animal tissues, 38 ; 
insects, 138 ; diatomacess, 144 ; 
I vegetable sections, 141. 

Preservation of vegetable tissues, 
131. 

Prussian blue injection mass, 64. 

Prussian blue staining fluid, 66, 
67, 68. 

Pseudoscopic vision, 25. 



E. 

RADIOLARIA, on cleaning and 

mounting, 157. 
Eat, 60. 
Beagents, constitution and action 

of, 4. 

Bed (injection) mass, 61, 62, 63. 
Eetina of eye, 16, 18. 
Butherford's microtome, 121. 



S. 



SAFFRANIN, 91. 
Salt solution, 53. 
Sclerotic coat of eye, 15, 18. 
Sciatic nerve, 58. 
Section cutting, 119. 
Septum cysternse lymphaticse, 56. 
Shellac cement, 107. 
Shells, to clean diatoms growing 
on, 146. 



INDEX. 



207 



Silver nitrate, 55. 

Skin, 60. 

Spiller's purple, 91. 

Sguamous epithelium, 54. 

Staining fluids (on), 77 ; anilin, 
87 ; anilin blue black, 86 : car- 
mine, 82, 83 ; haematoxylin, 79, 
80, 81; picro-carmine, 84, 85; 
sulph-indigotate of soda, 85. 

Staining, triple, 87. 

Staining (on), 77 ; with anilin dyes, 
88 ; 89, 90, 91 ; bacilli and bac- 
teria, 91, 92, 93 ; picro-carmine, 
144. 

Staining and mounting vegetable 
sections, 143. 

Stereoscopic vision, 25. 

Striped muscle, 60. 

Styrax, 105. 

Sulph-indigotate of soda, 85. 

Syringe, injecting with, 71 ; 
Higginson's, 75, 76. 



T. 



TAIL of mouse, 60; rat, 60. 
Tendon, 57, 60. 
Thoma microtome, the, 129. 
Thompson's, Rev. Gordon, process 

for staining and mounting 

bacilli, &c., 94. 
Transitional epithelium, 54. 



Triple staining, 87. 

Turpentine, 97 ; clearing with, 97. 

V. 

VABNISH, asphalt, 107. 

Vegetable tissues, preservation of, 
131 ; sections, preparation of, 
141 ; staining and mounting, 
142. 

Vesuvin, 91. 

Vision in man, limited, 10. 

Vitreous humour of eye, 16. 

W. 

WATER, 54. 

Wax and oil, embedding in, 113. 

Weigert's hsematoxylin staining 

fluid, 80 ; picro-carmine, 84 ; 

for bacilli, 94. 
Weights, French, conversion into 

English ditto, 37. 
White zinc cement, 107. 
Williams' freezing microtome, 

123. 
Woodhead, Dr. ^Sims, injection 

mass, 63. 

X. 

XYLOL balsam, 78, 88, 96. 
Xylol and phenol clearing medium, 
88, 95. 



DOLLOND & CO., 

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ESTABLISHED 1750. 



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The S.W. Histological and Botanists' Microscope as above Fig. With 
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This instrument is confidently recommended to Students as thoroughly 
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IR,. GK IMI.A_SO:Er 

(From J. SWIFT), 

69, PARK ROAD, CLAPHAM, LONDON, S.W. 



ESTABLISHED 1880. 



Living Specimens for the Microscope. 

JOHN HOOD, F.E.M.S., 

BEGS to intimate to Teachers and Students of Biology, and those studying 
Natural History with the aid of the Microscope, that he has always at 
hand the following list of Pond Life: 

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MICROSCOPE Stanley's New Histological, 

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I Back of Title 
) 


ANATOMY 






9, etc. 


ART, ARTISTIC ANATOMY, ETC. 






11, etc. 


CHEMISTRY 






14, etc. 


MEDICINE, SURGERY, AND ALLIED SCI 


ENCES 




26, etc. 


PHARMACY 






30, etc. 


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ALPHABETICAL INDEX OF AUTHORS. 



ABERCROMBIE (J.) On Tetany in Young Children 15 

ADAMS (W.) Surgical Treatmeni of Deformities 17 

ALLAN (F. J.) Aids to Sanitary Science 31 

ALLAN (J. H.) Tables of Doses 25 

ALLEN (Alfred) Microscopical Science : wrapper 

ALLINGHAM (H. W.) Colotomy 8 

ATTENDANTS. Handbook for Attendants on the Insane 24 

BAKER (Benson) How to Feed an Infant 28 

BALL Nose and Pharynx 28 

BAN HAM Veterinary Posological Tables 39 

BANNATYNE (A.) Aids to Pathology 29 

BEACH (Fletcher) Psychological Medicine 31 

BERNARD (Claude) and HUETTE'S Text-book of Operative Surgery 33 

BLACK (C). Atlas of the Male Organs of Generation 10 

BLACKLEY (C. H.) Hay Fever, its Causes and Treatment 22 

BODDY (E. M.) History of Salt 32 

Hydropathy 23 

BOWDICH (Mrs.) Confidential Chats with Mothers 15 

BOWLES (R. L. ) On Stertor and Apoplexy u 

BO YD (Stanley) Movable Atlas of the Foot, its Bones and Muscles 21 

BRAND (A. T.) Pocket Case Book 14 

BROCHARD (J.) Practical Guide for the Young Mother 28 

BROWN (George) The Student's Case-book 14 

Aids to Anatomy 9 

Aids to Surgery 33 

BROWNE (Lennox) The Throat and Nose, and their Diseases 34 

Diphtheria 18 

BROWNE ( W. J.) The Moon, its Influence on Weather 27 

BURTON (J. E.) Translation of Ebstein's Gout 22 

CAMERON (Chas.) Microbes in Fermentation, Putrefaction, and Disease ... 12 

The Cholera Microbe and How to Meet It 15 

CAMERON (Sir C. A.) History of the Royal College of Surgeons in Ireland 23 

CAMPBELL (C. M.) Skin Diseases of Infancy and Early Life 32 

CANTLIE (Jas.) Atlas of the Hand 10 

Text-book of Naked- Eye Anatomy 9 

C ARD WELL (B.) Translation of Hygiene of Beauty 24 

CASSFLLLS (J. Patterson) Deaf-mutism and the Education of the Deaf-mute 17 

CHARCOT (J. M.) Bright's Disease of the Kidneys 25 

CHRISTY (T.) Dictionary of Materia Medica 25 

CHURCHILL (Fleetwood) Obstetrical and Gynaecological Nursing 28 

CLARKE (J. Jackson) Cancer, Sarcoma and other Morbid Growths 14 

CLARKE (Percy) Medical Laws 26 

CLARKE (E. H.) The Building of a Brain 13 

CLARKE (Ernest) Atlas of Eye 20 

COFFIN (R. J. Maitland) Obstetrics 28 

COLE (M. J.) Modern Microscopy ... 27 

COOMBE (Russell) Epitome of B. P. 30 

CO O PER (R. T.) On Vascular Deafness 18 

COTTERELL (Ed.) The Pocket Gray, or Anatomist's Vade Mecum 9 

COURTENAY (E.) Practice of Veterinary Medicine 39 

COZZOLINO (V.) The Hygiene of the Ear 18 

CRAWFORD (W. S.) Ulcers and their Treatment 35 

CROOKE (G. F.) The Pathology of Tuberculosis 17 

CROSS (M. J.) Modern Microscopy 27 

CRUISE (F. R.) Hydropathy 23 

CULLIMORE (D. H.) Consumption as a Contagious Disease 16 



2 Baillifere, Tindall, and Cox's Books. 

PAGE 

CULLIMORE (D. H.) The Book of Climates 16 

DARLING (W.) Anatomography, or Graphic Anatomy 9 

The Essentials of Anatomy 9 

DAWSON (W. E.) Guide to the Examinations of the Apothecaries' Society 19 

DAY (W. H.) Irritable Brain in Children 13 

DENNIS ( Hy. J. ) Second-Grade Perspective Drawing 1 1 

Third-Grade Perspective Drawing 12 

DESSAR (L. A.) Catarrhs and Colds 14 

DOLAN (T. M.) Whooping Cough, its Pathology and Treatment 35 

DOWSE (T. Stretch) Apoplexy 11 

Syphilis of the Brain and Spinal Cord 13 

Skin Diseases from Nervous Affections 32 

The Brain and the Nerves and Influenza 13 

DRAGENDORFF (Prof. G.) Plant Analysis 15 

DRYSDALE (C. R.) Nature and Treatment of Syphilis 34 

DRYSDALE (John) The Protoplasmic Theory of Life 34 

DUDGEON (R. E.) The Sphygmograph 32 

DUFFEY (G. F.) Note-taking 14 

EBSTEIN (Prof.) The Treatment of Gout 22 

EDWARDS (F. Swinford) Urinary Surgery 35 

ERSKINE (T.) Hygiene of the Ear 19 

EVANS (C.'W. De Lacy) How to Prolong Life? 18 

Consumption : its Causes, Treatment, etc 16 

E WART (W.) Cardiac Outlines 14 

Heart-Studies, Chiefly Clinical 22 

How to Feel the Pulse 32 

Symptoms and Physical Signs 14 

FAU (}.) Artistic Anatomy of the Human Body II 

Anatomy of the External Form of Man II 

FIELD (G. P.) Diseases of the Ear 18 

FINNY (F. M.) Clinical Fever Chart 21 

FITZGERALD (H. P.) Dictionary of British Plants and Flowers 13 

FLAXMAN (J.) Elementary Anatomical Studies for Artists H 

FLEMING (G.) Text-book of Veterinary Obstetrics 39 

Neumann's Parasites of Domestic Animals 39 

Text-book of Veterinary Surgery 39 

Roaring in Horses 40 

Practical Horse-Shoeing 40 

Animal Plagues, their History, Nature and Treatment 40 

Contagious Diseases of Animals 40 

Tuberculosis 40 

Human and Animal Variolse 40 

Heredity and Contagion in the Propagation of Tuberculosis 40 

FORD Ophthalmic Notes 20 

FOTHERGILL (J. Milner) Chronic Bronchitis 13 

The Physiological Factor in Diagnosis 17 

Aids to Diagnosis 18 

The Physiologist in the Household 31 

Diseases of Sedentary and Advanced Life 29 

Aids to Rational Therapeutics 34 

Vaso-Renal Changes 24 

FOY (Geo.) Anaesthetics : Ancient and Modern 9 

FUCHS (Dr.) The Causes and Prevention of Blindness 20 

GANT (F. J.) Text-book of the Science and Practice of Surgery 33 



Baillifere, Tindall, and Cox's Books. 



GANT (F. J.) Diseases of the Bladder, Prostate Gland, and Urethra 13 

Examinations by the Conjoint Board 19 

Students' Surgery 33 

GARMANY (J. J.) Surgery on the Cadaver 33 

GARROD (A. E.) Handbook of Medical Pathology 29 

GEMMELL (Wm.) Derraic Memoranda 32 

GERSTER Aseptic and Antiseptic Surgery 33 

GIRAUD-TEULON Anomalies of Vision 20 

GLASGOW-PATTESON (R.) Skin and Hair 32 

GOOD ALL (E.) Microscopical Examinations of the Brain 23 

GORDON (Chas. A.) Our Trip to Burmah 13 

Aids to Psychological Medicine 38 

Life on the Gold Coast 8 

Lessons in Military Hygiene and Surgery 23 

A Manual of Sanitation 23 

Rabies and Hydrophobia 23 

Island of Madeira 16 

GORDON (T. Hurd) Aids to Practical Chemistry 36 

GORE (Albert A.) Our Services Under the Crown 27 

Medical History of African Campaigns 8 

GOULD Illustrated Dictionary of Medicine, Biology, etc 18 

GOW (W. J.) Handbook of Medical Pathology 29 

GRANVILLE (Mortimer) Gout 22 

GREEN (F. W. Edridge) Memory 27 

Detection of Colour Blindness.. 20 

GREENWOOD (J.) Laws Affecting Medical Men 26 

GREENWOOD (Major) Aids to Zoology 35 

GRESSWELL (J. B. and A. G. ) Manual of Equine Medicine and other works 40 

GREVILLE (H. Leicester) Student's Hand-book of Chemistry 15 

GRIFFITHS (A. B.) Micro-Organisms 12 

GRIFFITHS (W. H.) Text-book of Materia Medica and Pharmacy 25 

Notes for Pharmacopoeial Preparation . 30 

Posological Tables 31 

GUBB (Alfred S.) Aids to Gynaecology 22 

GUILLEMARD (F. H. H.) Endemic Hsematuria 20 

HAIG-BROWN Tonsillitis 35 

HALTON (R. J.) Short Lectures on Sanitary Subjects 24 

HANDBOOK for Attendants on the Insane 28 

HARRIS (Vincent) Manual for the Physiological Laboratory 23 

HARRIS (V. D.) Kuhne's Guide to the Demonstration of Bacteria 12 

HARTMANN (Prof.) On Deaf-mutism, Translation by Dr. Cassells 17 

HAYNES (Stanley) Healthy Homes 24 

HAZARD (W. P.) Diseases of Live Stock 41 

HE1BERG (Jacob) Atlas of Cutaneous Nerve Supply 27 

HEPPEL Analytical Conic Sections 38 

HERRINGHAM (W. P.) Handbook of Medical Pathology 29 

HERSCHELL (Geo.) Indigestion 24 

Heart Diagrams and Case-book 22 

HEWITT (Frederic) Anaesthetics 9 

HILL (J. W.) Principles and Practice of Bovine Medicine 40 

Management and Diseases of the Dog 40 

HIME (T. W.) Cholera: Ho\v to Prevent and Resist It 15 

The Practical Guide to the Public Health Acts 31 

HOARE Veterinary Therapeutics 40 

HOGG (Jabez) The Cure of Cataract 20 



4 Bailliere, Tindall, and Cox's Books. 

PAGE 

HOGG (Jabez) The Impairment of Vision from Shock 20 

Parasitic or Germ Theory of Disease 12 

HOPGOOD (T. F.) Notes on Surgical Treatment 33 

HORNER (Professor) On Spectacles 20 

HOWAT (G. R.) How to Prevent and Treat Consumption 16 

HUNTER (Ch.) Manual for Dental Laboratory 17 

HUSBAND (H. Aubrey) Handbook of Forensic Medicine 21 

Handbook of the Practice of Medicine 26 

Student's Pocket Prescriber 31 

Urine 35 



HUTCH INSON (Jonathan) Aids to Ophthalmic Medicine and Surgery 20 

ILLINGWORTH Hydrophobia 23 

INCE (J.) Latin Grammar of Pharmacy 30 

INTERNATIONAL MEDICAL CONGRESS 24 

JAMES (Brindley) Replies to Questions in Therapeutics 38 

JAMES (M. P.) Therapeutics of the Respiratory Passages 34 

Vichy and its Therapeutical Resources 35 

JENNINGS (C. E.) On Transfusion of the Blood and Saline Fluids 35 

Cancer and its Complications 14 

JENNINGS (Oscar) On the Cure of the Morphia Habit 27 

JESSETT (F. B.) Surgical Diseases of Stomach and Intestines 8 

Cancer of the Mouth and Tongue 14 

Cancer of the Uterus 14 

JONES (H. Macnaughton) The Diseases of Women 22 

Subjective Noises in the Head and Ears 18 

Hints for Midwives 28 

and STEWART Handbook of Diseases of the Ear and Naso- 

Pharynx 19 

JONES (H.) Guide to Sanitary Science Exams 32 

JONES (T. Wharton) Blood in Inflammation 24 

JUKES-BROWNE (A. J.) Palaeontology (in Penning's Field Geology) 21 

KAST AND RUMPEL Illustrations of Pathological Anatomy 29 

KEETLEY(C.R. B.) Guide to the Medical Profession 26 

Surgery of Knee Joint 33 

KENNEDY (Hy.) An Essay on Fatty Heart , 22 

KINGZETT Nature's Hygiene 23 

KNIGHT (G. D.) Movable Kidney 25 

KUHNE Demonstration of Bacteria 12 

LAMBERT (J.) The Germ Theory of Disease 40 

LEASK (J. G.) Questions at Medical Science Examinations 19 

LEDWICH (J.) Anatomy of Inguinal and Femoral Regions 9 

LEONARD (H.) The Pocket Anatomist 9 

Bandaging 13 

Hair 22 

and CHRISTY Dictionary of Materia Medica 25 

LE SUEUR Analytical Geometry, Straight Line and Circle 38 

LETHEBY(Hy.) The Sewage Question 32 

LIAUTARD (A.) Animal Castration 40 

Diseases of Live Stock 41 

Lameness of Horses 40 

Operative Veterinary Surgery 40 

LITHGOW (R. A . Douglas) From Generation to Generation 22 

LOWNE (B. T.) Aids to Physiology ., 37 

LUNN(C.) The Philosophy of Voice 35 



Bailli&re, Tindall, and Cox's Books. 5 

PAGE 

LUNN (C.) Artistic Voice in Speech and Song 35 

LUPTON (J. I.) Horses : Sound and Unsound 40 

MACDOUGALL (A. M.) The Maybrick Case 21 

MACKENZIE (Sir M.) Diseases of the Throat (in Gant's Surgery) 33 

McCAW (John) Aids to the Diagnosis and Treatment of Diseases of Children 15 

MADDEN (T. More) Clinical Gynaecology 22 

Churchill's Obstetrical Nursing 28 

MADDICK (Distin) Stricture of the Urethra 33 

MAGNE (Dr.) How to Preserve the Sight 20 

MARTIN (B. R.) Diphtheria 18 

MARTIN (J. W. & J.) Ambulance Work (Questions and Answers) 8 

Nursing (Questions and Answers) 28 

MASSE (J. N.) Text-book of Naked-Eye Anatomy 9 

MAYBURY Student's Chemistry 15 

McARDLE (J. S.) Notes on Materia Medica 26 

McBRIDE Anatomical Outlines of the Horse 41 

McLACHLAN (John) Anatomy of Surgery 33 

MEARS (W. P.) Schematic Anatomy ...'. 9 

MELDON (Austin) A Treatise on Gout 22 

MEYRICK (J. J.) Stable Management in India .' 41 

MILLARD (H. B.) Bright's Disease of the Kidneys 25 

MILLER (B. E.) Diseases of Live Stock 41 

MOLONY (M. J.) Rupture of the Perineum 32 

MONIN (E.) Hygiene of Beauty 24 

MOORE (E. H.) Clinical Chart for Hospital and Private Practice 34 

MOORE (J. W.) Text Book of Eruptive and Continued Fevers 21 

MORDHORST (Carl) Rheumatism. Its Treatment by Electric Massage ... 32 

MORGAN (John) The Dangers of Chloroform and Safety of Ether 8 

MORRIS (Malcolm) The Skin (in Gant's Surgery) 33 

MUCKLEY (W. J.) Student's Manual of Artistic Anatomy II 

A Handbook for Painters and Art Students on the Use of Colours 16 

MURRAY (R. Milne) Pregnancy 10 

MURRELL (W.) Aids to Forensic Medicine and Toxicology 21 

Prevention of Consumption 16 

MUTER (J.) Manual of Analytical Chemistry 15 

NALL (S.) Aids to Obstetrics 28 

NAPHEYS (G. H.) Handbook of Popular Medicine 18 

Modern Therapeutics 34 

NATIONAL SOCIETY FOR PREVENTION OF BLINDNESS 20 

NEUMANN (L. G.) Treatise on Parasites and Parasitic Diseases of Domes- 
ticated Animals 39 

NORTON (A. T.) Text-book of Operative Surgery 33 

Osteology for Students 23 

Affections of the Throat and Larynx 35 

Movable Atlas of the Skeleton 10 

Clinical Lectures on Recent Surgery 33 

OGSTON On Unrecognised Lesions of the Labyrinth 18 

ORMSBY (L. H.) Deformities of the Human Body 17 

Phimosis and Paraphimosis 30 

PALFREY (J.) Atlas of the Female Organs of Generation 18 

PALMER (J. F.) How to Bring up Children by Hand 28 

PARKE (Surgeon) Climate of Africa (in Cullimore's Book of Climates) 16 

PEDDIE (W.) Manual of Physics 30 

PENNING (W. H.) Text-book of Field Geology 21 



6 Bailliere, Tindall, and Cox's Books. 



PENNING (W. H.) Engineering Geology 21 

Notes on Nuisances, Drains, and Dwellings ... 24 

PETTENKOFER (Von) Cholera : How to Prevent and Resist It 15 

PIERSOL (G. A.) Text-book of Normal Histology 23 

POLITZER (Prof.) Dissections of the Human Ear 19 

Text-Book of Diseases of the Ear 19 

POWER (Hy.) Movable Atlas of the Eye, and the Mechanism of Vision 10 

Diseases of the Eye (in Gant's Surgery) 33 

POWER (D'Arcy) Handbook for the Physiological Laboratory 23 

POYSER (R.) Stable Management of Troop Horses in India 41 

PRATT (W.) A Physician's Sermon to Young Men 27 

PROCTOR (Richd.) The Stars and the Earth 12 

PSYCHOLOGICAL ASSOCIATION'S Handbook for Attendants on the 

Insane 28 

PURVES (L.) Aural Diseases (in Gant's Surgery) 33 

RABAGLIATI (A.) Muscular Affections which Simulate Diseases of the 

Pelvic Organs in Women 22 

The Classification and Nomenclature of Diseases 1 8 

REGIS Mental Medicine 27 

REMSEN (Ira) Principles of Theoretical Chemistry 15 

RENTOUL Reform of Medical Charities 26 

REYNOLDS (R. S.) The Breeding and Management of Draught Horses 41 

RICHARDS (J. M.) A Chronology of Medicine 26 

RICHARDSON (B. W.) The Healthy Manufacture of Bread 21 

RI VINGTON ( W. ) Medical Education and Organization 26 

ROBERTSON (William) A Handbook of the Practice of Equine Medicine... 41 

ROCHE (J.) Hernia and Intestinal Obstruction 23 

ROCHET (Chas.) The Prototype of Man, for Artists 12 

ROSE (W.) Neuralgia 28 

ROTH (W. E.) Elements of School Hygiene 23 

Theatre Hygiene 23 

ROUTH (C. H . F. ) Overwork and Premature Mental Decay 29 

On Checks to Population 31 

RUFFER (Armand) Illustrations of Pathological Anatomy 29 

SARCEY (F.) Mind your Eyes 20 

SCHOFIELD (A. T.) Examination Cards Pathology 20 

Minor Surgery and Bandaging 33 

SEMPLE (C. E. A.) Aids to Botany 13 

Aids to Chemistry 14 

Aids to Materia Medica 25 

Aids to Medicine 26 

Aids to Pharmacy 30 

- Diseases of Children 15 

The Voice Musically and Medically Considered 35 

The Pocket Pharmacopoeia 30 

SEWILL(Hy.) Manual of Dental Surgery 17 

Dental Caries and the Prevention of Dental Caries 17 

SHARMAN (J. S.) Notes on Inorganic Materia Medica 26 

SIMON (W.) A Manual of Chemistry 15 

SMITH (F. A. A.) Keep your Mouth Shut 32 

SMITH (F.) Manual of Veterinary Hygiene 41 

Manual of Veterinary Physiology 41 

SOHN (C. E. ) Dictionary of the Active Principles of Plants 15 

SPARKES (John C. L.) Artistic Anatomy n 

SQUIRE (P. W.) Posological Tables 31 



Bailliere, Tindall, and Cox's Books. 7 

PAGE 

STARK (A. Campbell) Practical Pharmacy 20, 30 

STARR (M. Allen) Brain Surgery 33 

STEPHENSON (J. B.) Medicinal Remedies 26 

STEVENS (Geo. T.) Nervous Diseases 27 

STEWART (W. R. H.) Practitioner's Handbook of Diseases of the Ear 19 

Aids to Otology 18 

STONE (G.) Translation of Politzer's Dissections of the Human Ear 19 

STRAHAN (J.) Extra-Uterine Pregnancy 28 

STUDENTS' AIDS SERIES 36 

SUTTON (H. G.) Lectures on Medical Pathology 29 

SUTTON (Bland) Dermoids 17 

SWEETING (R. D. R.) The Sanitation of Public Institutions 24 

SYMINGTON (J.) Anatomy of the Child 9 

TELLOR(L. V.) Diseases of Live Stock 41 

TEULON (G.) The Functions of Vision 20 

THIN (George) Introduction to Practical Histology 23 

THOMAS Hydatid Disease 23 

THOROWGOOD ( J. C.) Consumption ; its Treatment by the Hypophosphites 16 

The Treatment of Bronchial Asthma 12 

Aids to Physical Diagnosis 18 

THUDICHUM (J. L. W.) The Physiological Chemistry of the Brain 13 

Aids to Physiological Chemistry 38 

Aids to Public Health 31 

Polypus in the Nose 31 

The Coca of Peru, and its Remedial Principles 16 

The Spirit of Cookery 21 

TICHBORNE (Professor) The Mineral Waters of Europe 27 

TIDY (Meymott) and CLARKE (Percy) Medical Laws 26 

TIM MS (G.) Consumption ; its Nature and Treatment 16 

Alcohol in some Clinical Aspects, a Remedy, a Poison 8 

TOMSON Medical Electricity 19 

TRANSACTIONS of Royal Academy of Medicine in Ireland Inside cover 

TUCKEY (C. Lloyd) Psycho-Therapeutics 24 

TURNER (Dawson) Manual of Medical Electricity 19 

TYSON (J.) The Urine, a Guide to its Practical Examination 35 

UNDERWOOD (Arthur S.) Aids to Dental Surgery 17 

Aids to Dental Histology 17 

USHER (J. E.) Alcoholism 8 

VINTRAS Diabetes 18 

WAGSTAFFE (W. W.) Atlas of Cutaneous Nerve Supply 27 

WALDO and WALSH Bread, Bakehouses 12 

WALLACE (J.) Localised Peritonitis 29 

WALSH (D.) Aids to Examinations 19 

WALSHAM Deformities of the Foot 21 

WALSHAM and POWER Surgical Pathology 33 

WELPLY (J. J. ) Creameries and Infectious Diseases 24 

WHERRY (Geo.) Clinical Notes on Nerve Disorders 27 

WILLIAMS (J. W.) Aids to Biology 13 

WILLIAMSON (J. M.) Ventnor and the Undercliff 16 

WILLSON (A. Rivers) Chemical Notes for Pharmaceutical Students i; 

WILSON (J.) A Manual of Naval Hygiene 24 

WINDLE (B. C. A.) Proportions of the Human Body 12 

WINSLOW (L. S. Forbes) Fasting and Feeding 20 

WITKOWSKI (G. J.) Movable Atlases of the Human Body 10 

YONGE (E. S.) Aids to Surgical Anatomy 36 

YOUNG (J. K.) Orthopedic Surgery 21 



Baillifere, Tindall, and Cox's Books. 



AN 

ALPHABETICAL INDEX OF WORKS, 

IN 
MEDICINE, SURGERY, SCIENCE AND ART, 

PUBLISHED BY 

BAILLIERE, TINDALL, & COX, 



Abdominal Surgery. Colotomy, Inguinal, Lumbar or Transverse ; 
for Cancer, or Stricture with Ulceration, of the large Intestine. 
By HERBERT W. ALLINGHAM, F.R.C.S., Surgeon to the Great 
Northern Hospital, Assistant Surgeon to St. Mark's Hospital 
for Diseases of the Rectum, Surgical Registrar to St. George's 
Hospital. With six plates and numerous illustrations. Price 6s. 

Abdominal Surgery. The Surgical Diseases and Injuries of the 
Stomach and Intestines. By F. BOWREMAN JESSETT, F.R.C.S. 
Eng., Surgeon to the Cancer Hospital. Copiously illustrated. 
Price 7s. 6d. 

Africa. A Contribution to the Medical History of our West 
African Campaigns. By Surgeon-Major ALBERT A. GORE, M.D., 
Sanitary Officer on the Staff. Price 10s. 6d. 

Africa. Life on the Gold Coast. A Description of the Inhabitants, 
their Modes and Habits of Life ; Hints to Travellers and others 
in Western Africa. By Surgeon-General GORDON, M.D., C.B., 
Hon. Physician to the Queen. Price 2s. 6d. 

Alcohol, in some Clinical Aspects : A Remedy, a Poison. By 
GODWIN TIMMS, M.D., M.R.C.P. Lond., Senior Physician to 
the North London Consumption Hospital. Price Is. 

Alcoholism and its Treatment. By JOHN E. USHER, M.D., 
F.R.G.S. Price 3s. 6d. 

" Will be found interesting and suggestive." The. Times. 

" A very full account of the methods of treating the disease of inebriety is contained in this 
interesting work." British Medical Journal. 

Ambulance Work. Questions and Answers on " First Aid to the 
Injured." By JOHN W. MARTIN, MD., and JOHN MARTIN, 
F.R.C.S. Twentieth thousand. Price Is. net. 

Anaesthetics. The Dangers of Chloroform and the Safety and 
Efficiency of Ether in Surgical Operations. By JOHN MORGAN, 
M.D., F.R.C.S. Second thousand, price 2s: 



Baillifere, Tindall, and Cox's Books. 9 

Anaesthetics. Selected Methods in the Administration of Nitrous 
Oxide and Ether. By FREDERIC HEWITT, M.A., M.D. Cantab., 
Lecturer on Anaesthetics at the London Hospital. Price 2s. 6d. 

Anaesthetics : Ancient and Modern. Their Physiological Action, 
Therapeutic Use, and Mode of Action. By GEORGE FOY, 
F.R.C.S., Surgeon to the Richmond Hospital Price 3s. 6d. net. 

Anatomography ; or, Graphic Anatomy. A new method of 
grasping and committing to memory the most difficult points 
required of the student. By W. DARLING, M.D., F.R.C.S. Eng., 
Professor of Anatomy in the University of New York. Price Is. 

Anatomy. Aids to Anatomy. By GEORGE BROWN, M.R.C.S., and 
P. MACLEOD YEARSLEY, F.R.C.S. Price 2s. 6d. cloth, 2s. sewn. 

Anatomy. Text-Book of Naked-Eye Anatomy. With 113 Steel 
Plates, designed under the direction of Professor MASSE. Text by 
JAS. CANTLIE, M.B., C.M. (Honours), F.R.C.S., Charing Cross 
Hospital. Third edition. Plain, 25s., coloured, 50s., half calf. 

Anatomy. The Essentials of Anatomy. A Text-book for Students 
and a book of easy reference to the Practitioner. By W. 
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Baillifere, Tindall, and Cox s Books. 17 



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18 Baillifere, Tindall, and Cox's Books. 



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Baillifere, Tindall, and Cox's Books. 19 

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20 Baillifere, Tindall, and Cox's Books. 



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22 Baillifere, Tindall, and Cox's Books. 

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26 Baillifere, Tindall, and Cox's Books. 



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Baillifere, Tindall, and Cox s Books. 27 

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28 Baillikre, Tindall, and Cox's Books. 

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Baillike, Tindall, and Cox's Books. 29 

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Bailliere, Tindall, and Cox's Books. 31 

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32 Baillifere, Tindall, and Cox's Books. 

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Bailliere, Tindall, arid Cox's Books. 33 

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34 Baillikre, Tindall, and Cox's Books. 



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Throat. Movable Atlas of the Throat, and the Mechanism of Voice, 
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Baillifere, Tindall, and Cox's Books. 35 

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36 Bailliere, Tindall, and Cox's Books. 



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Bailliere, Tindall, and Cox's Books. 37 

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38 Bailliere, Tindall, and Cox's Books. 

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Bailliere, Tindall, and Cox's Books. 39 



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40 Baillifere, Tindall, and Cox's Books. 



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